The compounds of the present invention are antagonists of the VLA-4 integrin (xe2x80x9cvery late antigen-4xe2x80x9d; CD49d/CD29; or xcex14xcex21), the xcex14xcex27 integrin (LPAM-1 and xcex14xcex2p), and/or the xcex19xcex21 integrin, thereby blocking the binding of VLA-4 to its various ligands, such as VCAM-1 and regions of fibronectin, xcex14xcex27 to its various ligands, such as MadCAM-1, VCAM-1 and fibronectin, and/or xcex19xcex21 to its various ligands, such as tenascin, osteopontin and VCAM-1. Thus, these antagonists are useful in inhibiting cell adhesion processes including cell activation, migration, proliferation and differentiation. These antagonists are useful in the treatment, prevention and suppression of diseases mediated by VLA4-, xcex14xcex27-, and/or xcex19xcex21-binding and cell adhesion and activation, such as AIDS-related dementia, allergic conjunctivitis, allergic rhinitis, Alzheimer""s disease, aortic stenosis, asthma, atherosclerosis, autologous bone marrow transplantation, certain types of toxic and immune-based nephritis, contact dermal hypersensitivity, inflammatory bowel disease including ulcerative colitis and Crohn""s disease, inflammatory lung diseases, inflammatory sequelae of viral infections, meningitis, multiple sclerosis, myocarditis, organ transplantation, psoriasis, restenosis, retinitis, rheumatoid arthritis, septic arthritis, stroke, tumor metastasis, type I diabetes, vascular occlusion following angioplasty.
The present invention relates to oxygen and sulfur heterocyclic amide derivatives which are useful for the inhibition and prevention of leukocyte adhesion and leukocyte adhesion-mediated pathologies. This invention also relates to compositions containing such compounds and methods of treatment using such compounds.
Many physiological processes require that cells come into close contact with other cells and/or extracellular matrix. Such adhesion events may be required for cell activation, migration, proliferation and differentiation. Cell-cell and cell-matrix interactions are mediated through several families of cell adhesion molecules (CAMs) including the selecting, integrins, cadherins and immunoglobulins. CAMs play an essential role in both normal and pathophysiological processes. Therefore, the targetting of specific and relevant CAMs in certain disease conditions without interfering with normal cellular functions is essential for an effective and safe therapeutic agent that inhibits cell-cell and cell-matrix interactions.
The integrin superfamily is made up of structurally and functionally related glycoproteins consisting of a and b heterodimeric, transmembrane receptor molecules found in various combinations on nearly every mammalian cell type. (for reviews see: E. C. Butcher, Cell, 67, 1033 (1991); T. A. Springer, Cell, 76, 301 (1994); D. Cox et al., xe2x80x9cThe Pharmacology of the Integrins.xe2x80x9d Medicinal Research Rev. 14, 195 (1994) and V. W. Engleman et al., xe2x80x9cCell Adhesion Integrins as Pharmaceutical Targets.xe2x80x9d in Ann. Repts. in Medicinal Chemistry, Vol. 31, J. A. Bristol, Ed.; Acad. Press, NY, 1996, p. 191).
VLA-4 (xe2x80x9cvery late antigen-4xe2x80x9d; CD49d/CD29; or xcex14xcex21) is an integrin expressed on all leukocytes, except platelets and mature neutrophils, including dendritic cells and macrophage-like cells and is a key mediator of the cell-cell and cell-matrix interactions of these cell types (see M. E. Hemler, xe2x80x9cVLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.xe2x80x9d Ann. Rev. Immunol. 8, 365 (1990)). The ligands for VLA-4 include vascular cell adhesion molecule-1 (VCAM-1) and the CS-1 domain of fibronectin (FN). VCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells at sites of inflammation. (See R. Lobb et al. xe2x80x9cVascular Cell Adhesion Molecule 1.xe2x80x9d in Cellular and Molecular Mechanisms of Inflammation, C. G. Cochrane and M. A. Gimbrone, Eds.; Acad. Press, San Diego, 1993, p. 151.) VCAM-1 is produced by vascular endothelial cells in response to pro-inflammatory cytokines (See A. J. H. Gearing and W. Newman, xe2x80x9cCirculating adhesion molecules in disease.xe2x80x9d, Immunol. Today, 14, 506 (1993). The CS-1 domain is a 25 amino acid sequence that arises by alternative splicing within a region of fibronectin. (For a review, see R. O. Hynes xe2x80x9cFibronectins.xe2x80x9d, Springer-Velag, N.Y., 1990. ) A role for VLA-4/CS-1 interactions in inflammatory conditions has been proposed (see M. J. Elices, xe2x80x9cThe integrin xcex14xcex21 (VLA-4) as a therapeutic targetxe2x80x9d in Cell Adhesion and Human Disease, Ciba Found. Symp., John Wiley and Sons, NY, 1995, p. 79).
xcex14xcex27(also referred to as LPAM-1 and xcex14xcex2p) is an integrin expressed on leukocytes and is a key mediator of leukocyte trafficking and homing in the gastrointestinal tract (see C. M. Parker et al., Proc. Natl. Acad. Sci. USA, 89, 1924 (1992)). The ligands for xcex14xcex27 include mucosal addressing cell adhesion molecule-1 (MadCAM-1) and, upon activation of xcex14xcex27, VCAM-1 and fibronectin (Fn). MadCAM-1 is a member of the Ig superfamily and is expressed in vivo on endothelial cells of gut-associated mucosal tissues of the small and large intestine (xe2x80x9cPeyer""s Patchesxe2x80x9d) and lactating mammary glands. (See M. J. Briskin et al., Nature, 363, 461 (1993); A. Hamann et al., J. Immunol., 152, 3282 (1994)). MadCAM-1 can be induced in vitro by proinflammatory stimuli (See E. E. Sikorski et al. J. Immunol., 151, 5239 (1993)). MadCAM-1 is selectively expressed at sites of lymphocyte extravasation and specifically binds to the integrin, xcex14xcex27.
The xcex19xcex21 integrin is found on airway smooth muscle cells, non-intestinal epithelial cells (see Palmer et al., J. Cell Biol., 123, 1289 (1993)), and neutrophils, and,less so, on hepatocytes and basal keratinocytes (see Yokosaki et al., J. Biol. Chem., 269,24144 (1994)). Neutrophils, in particular, are intimately involved in acute inflammatory responses. Attenuation of neutrophil involvement and/or activation would have the effect of lessening the inflammation. Thus, inhibition of xcex19xcex21 binding to its respective ligands would be expected to have a positive effect in the treatment of acute inflammatory conditions.
Neutralizing anti-xcex14 antibodies or blocking peptides that inhibit the interaction between VLA-4 and/or xcex14xcex27 and their ligands have proven efficacious both prophylactically and therapeutically in several animal models of disease, including i) experimental allergic encephalomyelitis, a model of neuronal demyelination resembling multiple sclerosis (for example, see T. Yednock et al., xe2x80x9cPrevention of experimental autoimmune encephalomyelitis by antibodies against xcex14xcex21 integrin.xe2x80x9d Nature, 356, 63 (1993) and E. Keszthelyi et al., xe2x80x9cEvidence for a prolonged role of xcex14 integrin throughout active experimental allergic encephalomyelitis.xe2x80x9d Neurology, 47, 1053 (1996)); ii) bronchial hyperresponsiveness in sheep and guinea pigs as models for the various phases of asthma (for example, see W. M. Abraham et al., xe2x80x9cxcex14-Integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.xe2x80x9d J. Clin. Invest. 93, 776 (1993) and A. A. Y. Milne and P. P. Piper, xe2x80x9cRole of VLA-4 integrin in leucocyte recruitment and bronchial hyperresponsiveness in the guinea-pig.xe2x80x9d Eur. J. Pharmacol., 282, 243 (1995)); iii) adjuvant-induced arthritis in rats as a model of inflammatory arthritis (see C. Barbadillo et al., xe2x80x9cAnti-VLA-4 mAb prevents adjuvant arthritis in Lewis rats.xe2x80x9d Arthr. Rheuma. (Suppl.), 36 95 (1993) and D. Seiffge, xe2x80x9cProtective effects of monoclonal antibody to VLA-4 on leukocyte adhesion and course of disease in adjuvant arthritis in rats.xe2x80x9d J. Rheumatol., 23, 12 (1996)); iv) adoptive autoimmune diabetes in the NOD mouse (see J. L. Baron et al., xe2x80x9cThe pathogenesis of adoptive murine autoimmune diabetes requires an interaction between xcex14-integrins and vascular cell adhesion molecule-l.xe2x80x9d, J. Clin. Invest., 93, 1700 (1994), A. Jakubowski et al., xe2x80x9cVascular cell adhesion molecule-Ig fusion protein selectively targets activated xcex14-integrin receptors in vivo: Inhibition of autoimmune diabetes in an adoptive transfer model in nonobese diabetic mice.xe2x80x9d J. Immunol., 155, 938 (1995), and X. D. Yang et al., xe2x80x9cInvolvement of beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MadCAM-1) in the development of diabetes in nonobese diabetic micexe2x80x9d, Diabetes, 46, 1542 (1997)); v) cardiac allograft survival in mice as a model of organ transplantation (see M. Isobe et al., xe2x80x9cEffect of anti-VCAM-1 and anti-VLA-4 monoclonal antibodies on cardiac allograft survival and response to soluble antigens in mice.xe2x80x9d, Tranplant. Proc., 26, 867 (1994) and S. Molossi et al., xe2x80x9cBlockade of very late antigen-4 integrin binding to fibronectin with connecting segment-1 peptide reduces accelerated coronary arteripathy in rabbit cardiac allografts.xe2x80x9d J. Clin Invest., 95, 2601 (1995)); vi) spontaneous chronic colitis in cotton-top tamarins which resembles human ulcerative colitis, a form of inflammatory bowel disease (see D. K. Podolsky et al., xe2x80x9cAttenuation of colitis in the Cotton-top tamarin by anti-xcex14 integrin monoclonal antibody.xe2x80x9d, J. Clin. Invest., 92, 372 (1993)); vii) contact hypersensitivity models as: a model for skin allergic reactions (see T. A. Ferguson and T. S. Kupper, xe2x80x9cAntigen-independent processes in antigen-specific immunity.xe2x80x9d, J. Immunol., 150, 1172 (1993) and P. L. Chisholm et al., xe2x80x9cMonoclonal antibodies to the integrin a-4 subunit inhibit the murine contact hypersensitivity response.xe2x80x9d Eur. J. immunol., 23, 682 (1993)); viii) acute neurotoxic nephritis (see M. S. Mulligan et al., xe2x80x9cRequirements for leukocyte adhesion molecules in nephrotoxic nephritis.xe2x80x9d, J. Clin. Invest., 91, 577 (1993)); ix) tumor metastasis (for examples, see M. Edward, xe2x80x9cIntegrins and other adhesion molecules involved in melanocytic tumor progression.xe2x80x9d, Curr. Opin. Oncol., 7, 185 (1995)); x) experimental autoimmune thyroiditis (see R. W. McMurray et al., xe2x80x9cThe role of xcex14 integrin and intercellular adhesion molecule-1 (ICAM-1) in murine experimental autoimmune thyroiditis.xe2x80x9d Autoimmunity, 23, 9 (1996); and xi) ischemic tissue damage following arterial occlusion in rats (see F. Squadrito et al., xe2x80x9cLeukocyte integrin very late antigen-4/vascular cell adhesion molecule-1 adhesion pathway in splanchnic artery occlusion shock.xe2x80x9d Eur. J. Pharmacol., 318, 153 (1996; xii) inhibition of TH2 T-cell cytokine production including IL4 and IL-5 by VLA-4 antibodies which would attenuate allergic responses (J. Clinical Investigation 100, 3083 (1997). The primary mechanism of action of such antibodies appears to be the inhibition of lymphocyte and monocyte interactions with CAMs associated with components of the extracellular matrix, thereby limiting leukocyte migration to extravascular sites of injury or inflammation and/or limiting the priming and/or activation of leukocytes.
There is additional evidence supporting a possible role for VLA-4 interactions in other diseases, including rheumatoid arthritis; various melanomas, carcinomas, and sarcomas, including multiple myeloma; inflammatory lung disorders; acute respiratory distress syndrome (ARDS); pulmonary fibrosis; atherosclerotic plaque formation; restenosis; uveitis; and circulatory shock (for examples, see A. A. Postigo et al., xe2x80x9cThe xcex14xcex21/VCAM-1 adhesion pathway in physiology and disease.xe2x80x9d, Res. Immunol., 144, 723 (1994) and J.-X. Gao and A. C. Issekutz, xe2x80x9cExpression of VCAM-1 and VLA-4 dependent T-lymphocyte adhesion to dermal fibroblasts stimulated with proinflammatory cytokines.xe2x80x9d Immunol. 89, 375 (1996)).
At present, there is a humanized monoclonal antibody (Antegren(copyright), Athena Neurosciences/Elan) against VLA-4 in clinical development for the treatment of xe2x80x9cflaresxe2x80x9d associated with multiple sclerosis and a humanized monoclonal antibody (ACT-1(copyright)/LDP-02 LeukoSite) against xcex14xcex27 in clinical development for the treatment of inflammatory bowel disease. Several antagonists of VLA-4 and xcex14xcex27 have been described (D. Y. Jackson et al., xe2x80x9cPotent xcex14xcex21 peptide antagonists as potential anti-inflammatory agentsxe2x80x9d, J. Med. Chem., 40, 3359 (1997); H. N. Shroff et al., xe2x80x9cSmall peptide inhibitors of xcex14xcex27 mediated MadCAM-1 adhesion to lymphocytesxe2x80x9d, Bioorg. Med. Chem. Lett., 6, 2495 (1996); K. C. Lin et al., xe2x80x9cSelective, tight-binding inhibitors of integrin xcex14xcex21 that inhibit allergic airway responsesxe2x80x9d, J. Med. Chem., 42, 920 (1999); U.S. Pat. No. 5,510,332, WO97/03094, WO97/02289, WO96/40781, WO96/22966, WO96/20216, WO96/01644, WO96/06108, WO95/15973). There are reports of nonpeptidyl inhibitors of the ligands for xcex14-integrins (WO99/36393, WO98/58902, WO96/31206); A. J. Soures et al., Bioorg. Med. Chem. Lett., 8, 2297 (1998). There still remains a need for low molecular weight, specific inhibitors of VLA-4- and xcex14xcex27-dependent cell adhesion that have improved pharmacokinetic and pharmacodynamic properties such as oral bioavailability and significant duration of action. Such compounds would prove to be useful for the treatment, prevention or suppression of various pathologies mediated by VLA-4 and xcex14xcex27 binding and cell adhesion and activation.
PCT Application No. WO99/10312 discloses compounds having activity as inhibitors of binding between VCAM-1 and cells expressing VLA-4, and having the formula: 
PCT Application No. WO99/36393 discloses inhibitors of xcex14-mediated cell adhesion having the formula: 
PCT Application No. WO99/37618 discloses inhibitors of xcex14 integrins having the formula: 
wherein Het is a heteroaromatic ring and R is carboxylic acid.
U.S. Pat. No. 5,334,730 discloses diasteromeric carboxylic acid amides of the formulae 
These compounds are useful for the separation of optically active isomers of the heterocyclic carboxylic acid. No biological or therapeutic activity is disclosed for these compounds.
The present invention provides, in one aspect, a method for the prevention or treatment of diseases, disorders, conditions or symptoms mediated by cell adhesion in a mammal which comprises administering to said mammal an effective amount of a compound of Formula I: 
or a pharmaceutically acceptable salt thereof wherein:
A is a 4- to 8-membered monocyclic saturated heterocyclic ling having one to two heteroatoms chosen from O, S and S(O)m, optionally fused to an aryl group, wherein said heterocyclic ring and aryl group are optionally substituted with one to four substituents selected from a group independently selected from oxo, methylene and Rb;
Y is 1) a bond,
2) C1-10alkylene,
3) C2-10alkenylene,
4) C2-10alkynylene,
xe2x80x83wherein said alkylene, alkenylene and alkynylene are each optionally substituted with one to four substituents selected from Ra,
Z is 1) a bond, or
2) xe2x80x94C(R5)(R6)xe2x80x94
R1 is 1) hydrogen,
2) Cy,
3) OR5,
4) OC(O)R5;
5) OC(O)OR5,
6) OC(O)NRdRe,
7) SR5,
8) S(O)mR5,
9) SO2NRdRe,
10) C(O)R5,
11) C(O)OR5, 
12) C(O)NRdRe,
13) NRdRe,
14) NRdC(O)R5,
15) NRdC(O)OR5, 
16) NRdC(O)NRdRe,
17) NRdSO2R5;
xe2x80x83wherein Cy is optionally substituted with one to four substituents independently selected from Rb;
R2 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents independently selected from Ra;
R3 is
1) C1-10alkyl,
2) Ar1,
3) C1-10alkyl-Ar1,
4) Ar1xe2x80x94Ar2,
5) C1-10alkyl-Ar1xe2x80x94Ar2,
xe2x80x83wherein the alkyl group is optionally substituted with one to four substituents selected from Ra, and Ar1 and Ar2 are optionally substituted with one to four substituents independently selected from Rb,
R4 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl, wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents independently selected from Ra;
R5 is
1) hydrogen,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
5) Cy,
6) Cy-C1-10alkyl,
xe2x80x83wherein alkyl, alkenyl and alkynyl are optionally substituted with one to four substituents selected from Ra, and Cy is optionally substituted with one to four substituents independently selected from Rb;
R6 is
1) hydrogen,
2) a group selected from Rb;
Ra is
1) xe2x80x94ORd,
2) NRdS(O)mRe,
3) xe2x80x94NO2, 
4) halogen
5) xe2x80x94S(O)mRd,
6) xe2x80x94SRd,
7) xe2x80x94S(O)2ORd,
8) xe2x80x94S(O)mNRdRe,
9) xe2x80x94NRdRe,
10) xe2x80x94O(CRfRg)nNRdRe,
11) xe2x80x94C(O)Rd,
12) xe2x80x94CO2Rd,
13) xe2x80x94CO2(CRfRg)nCONRdRe,
14) xe2x80x94OC(O)Rd,
15) xe2x80x94CN,
16) xe2x80x94C(O)NRdRe,
17) xe2x80x94NRdC(O)Re,
18) xe2x80x94OC(O)NRdRe,
19) xe2x80x94NRdC(O)ORe,
20) xe2x80x94NRdC(O)NRdRe,
21) xe2x80x94CRd(N-ORe),
22) CF3, 
23) xe2x80x94OCF3,
24) C3-8cycloalkyl, or
25) heterocyclyl;
xe2x80x83wherein cycloalkyl and heterocyclyl are optionally substituted with one to four groups independently selected from oxo and Rc;
Rb is
1) a group selected from Ra,
2) C1-10alkyl,
3) C2-10alkenyl,
4) C2-10alkynyl,
5) Ar1,
6) C1-10alky-Ar1,
xe2x80x83wherein alkyl, alkenyl, alkynyl, and Ar1 are optionally substituted with one to four substituents selected from a group independently selected from Ra;
Rc is
1) halogen,
2) NRfRg,
3) C(O)ORf,
4) C1-4alkyl,
5) C1-4alkoxy,
6) aryl,
7) aryl C1-4alkyl,
8) hydroxy,
9) CF3,
10) OC(O)C1-4alkyl,
11) OC(O)NRfRg,
12) NRfC(O)Rg,
13) NRfSO2Rg, or
14) aryloxy;
xe2x80x83Rd and Re are independently selected from hydrogen, C1-10alkyl,
C2-10alkenyl, C2-10alkynyl, Cy and Cy C1-10alkyl, wherein alkyl, alkenyl, alkynyl and Cy are optionally substituted with one to four substituents independently selected from Rc; or
Rd and Re together with the atom(s) to which they are attached form a heterocyclic ring of 4 to 7 members containing 0-2 additional heteroatoms independently selected from oxygen, sulfur and Nxe2x80x94Rh;
Rf and Rg are independently selected from hydrogen, C1-10alkyl, Cy and Cyxe2x80x94C1-10alkyl; or
Rf and Rg together with the carbon to which they are attached form a ring of 5 to 7 members containing 0-2 heteroatoms independently selected from oxygen, sulfur and Nxe2x80x94Rh;
Rh is hydrogen, C1-10alkyl, or C(O)OC1-10alkyl;
Cy is selected from cycloalkyl, heterocyclyl and Ar1;
Ar1 and Ar2 are independently selected from aryl and heteroaryl;
m is 1 or 2;
n is an integer from 1 to 10.
In one embodiment said disease or disorder is selected from asthma, allergic rhinitis, multiple sclerosis, atherosclerosis, and inflammatory bowel disease.
In another aspect the present invention provides a method for preventing the action of VLA-4 in a mammal which comprises administering to said mammal a therapeutically effective amount of a compound of formula I.
Another aspect of the present invention provides a pharmaceutical composition which comprises a compound of formula I and a pharmaceutically acceptable carrier.
Another aspect of the present invention provides novel compounds of formula I, with the proviso that when R1xe2x80x94Y represents H, R4 is H, and R3 is (1) optionally substituted C1-10 alkyl, (2) phenyl unsubstituted or substituted with methyl, hydroxy, or methoxy, or (3) benzyl unsubstituted or substituted with methyl, hydroxy, or benzyl, then R5 is hydrogen, or a pharmaceutically acceptable salt thereof.
In one embodiment of compounds of formula I, A is 4- to 6-membered monocyclic saturated heterocyclic ring having one or two heteroatoms selected from O, S and S(O)m, and optionally substituted with one to four groups independently selected from oxo and Rb. Examples of suitable saturated heterocyclic ring include oxetane, thietane, tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, tetrahydrothiopyran, thioxane, dioxane. In another embodiment, A is aryl fused heterocyclic ring, and examples of which include dihydrobenzofuran, benzo-1,3-dioxolyl, and 1,2-dihydrothienyl. Examples of optional substituents for A include oxo, C1-10alkyl, C2-10alkenyl, Ar1, Ar1xe2x80x94C1-10alkyl, C1-10alkoxy, hydroxy, halogen, xe2x80x94S(O)2Rd, xe2x80x94S(O)2NRdRe, xe2x80x94NRdRe, xe2x80x94O(CRfRg)nNRdRe, xe2x80x94C(O)NRdRe, wherein alkyl, alkenyl, alkoxy, Ar1 are optionally substituted as provided under formula I. More particularly, suitable substituents for A include, but are not limited to, oxo, hydroxy, NRdRe such as amino, pyrrolidinyl, piperidinyl and morpholinyl, aryl such as phenyl and methoxyphenyl, C1-3alkyl-aryl such as benzyl, and C-5alkyl optionally substituted with a group selected from OC(O)NRdRe, NHC(O)Re, NHSO2Re, and ORd such as methyl, carbamoyloxymethyl such as 1-pyrrolidinylcarbonyloxymethyl, acylaminomethyl such as benzoylamino, sulfonylaminomethyl such as benzenesulfonylaminomethyl, and hydroxymethyl. Examples of suitable A include tetrahydrofuran-2-yl, 5-oxo-tetrahydrofuran-2-yl, 4-methyl-tetrahydrofuran-2-yl, 4-methylenetetrahydrofuran-2-yl, 4-hydroxymethyl-tetrahydrofuran-2-yl, 4-(pyrrolidinecarbonylmethyl)tetrahydrofuran-2-yl, 4-[(benzoylamino)methyl]-tetrahydrofuran-2-yl, 4-[(benzenesulfonylamino)methyl]tetrahydrofuran-2-yl, 3-oxo-5-methyltetrahydrofuran-2-yl, 3-oxo-5-benzyltetrahydrofuran-2-yl, 3-oxo-5-phenyltetrahydrofuran-2-yl, 3-hydroxy-5-phenyltetrahydrofuran-2-yl, 5-methyl-3-[(4-methoxy)phenyl]tetrahydrofuran-2-yl, 5-methyl-3-aminotetrahydrofuran-2-yl, 4-(1-pyrrolidinyl)tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, 2,2-dimethyl-5-oxo-tetrahydrofuran-3-yl, 2-tetrahydropyranyl, 2-tetrahydrothienyl-1,1-dioxide, 5-methyl-2-tetrahydrothienyl-1,1-dioxide, 2-thietanyl-1,1-dioxide, 2-thioxanyl-1,1-dioxide, methylisobenzofuranone, 2-dihydrobenzofuranyl, 2-benzo-1,3-oxolo-2-yl.
In another embodiment, Y is a bond or C1-5alkylene optionally substituted with one to two groups selected from Ra. Suitably, Y is a bond, methylene, ethylene, propylene, butylene, isopropylene, isobutylene, each optionally substituted with one to two groups selected from Ra, preferably hydroxy or C1-3alkoxy.
In another embodiment R1 is H, Cy optionally substituted with one to four substituents selected from Rb, OR5, OC(O)R5, NRdRe, C(O)NRdRe, C(O)OR5, C(O)NRdRe, NRdC(O)R5. Suitable substituents for Cy include halogen, alkyl, haloalkyl, alkoxy, amino, and carboxyl. Examples of suitable R1 are H, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 4-t-butylphenyl, 3-trifluoromethylphenyl, 3,5-bis(trifluoromethyl)phenyl, 3,4-dimethylphenyl, 2-thienyl, hydroxy, methoxy, acetoxy, amino, methylamino, morpholinocarbonyl, benzyloxycarbonyl, benzylaminocarbonyl, dimethylaminocarbonyl, dibenzylaminocarbonyl, pyrrolidinocarbonyl, piperazinocarbonyl, 4-methylpiperazinocarbonyl, ethylaminocarbonyl, acetylamino, N-methylacetylamino, cyclohexyl, 4-chloro-3-nitrophenhl, 3-nitrophenyl, 4-methoxyphenyl, 3,5-dimethoxyphenyl, benzoxazolyl, thiazolyl, 4-methylthiazolyl, and pyrrolidinyl.
In another embodiment, R3 is C1-5alkyl-Ar1 or C1-5alkyl-Ar1xe2x80x94Ar2, wherein Ar1 and Ar2 are optionally substituted with one to four groups independently selected from Rb. In a preferred subset R3 is C1-3alkyl-Ar1xe2x80x94Ar2 optionally substituted with one to four groups independently selected from Rb. More preferably R3 is CH2xe2x80x94Ar1xe2x80x94Ar2 wherein at least one Ar is a phenyl, and wherein Ar1 and Ar2 are optionally substituted with one or two groups selected from Rb. A particularly preferred group of R3 is substituted biphenylmethyl wherein one of the substituent(s) is located at the 2xe2x80x2-position of the biphenyl ring. Suitable Rb substituents for Ar are hydroxy, alkoxy, cycloalkoxy, cyano, amino, carbamyloxy, alkoxycarbonyl, carboxy, aminocarbonyl, alkoxy-alkoxy, and halogen. Examples of R3 include phenyl, benzyl, phenethyl, biphenylmethyl, 4-(1-morpholinocarbonyloxy)phenylmethyl, 4-(5-[1,3-dimethyl-2,4-pyrimidinedione])phenylmethyl, 4-(2-tert-butyloxycarbonylethyl)phenylmethyl, 4-(ethoxyethyl)phenylmethyl, 4-(hydroxymethyl)phenylmethyl, 4-(methoxymethyl)phenylmethyl, 3-(t-butoxy)phenylmethyl, 3-(t-butoxycarbonylmethoxy)phenylmethyl, 4-(1-pyrrolidincarbonyloxymethyl)phenylmethyl, 3-(2-ethoxyethoxy)phenylmethyl, 3-(2-methoxyethoxy)phenylmethyl, 3-(1-pyrrolidinecarbonyloxy)phenylmethyl, 4-(4xe2x80x2-methoxyphenoxy)phenylmethyl, 4-(3xe2x80x2-methoxyphenoxy)phenylmethyl, 4-(2xe2x80x2-methoxyphenoxy)phenylmethyl, 4-(1-pyrid-2(1H)one)phenylmethyl, 4-(2,6-dichlorobenzamido)phenylmethyl, 4-(4-(2xe2x80x2-cyano)biphenylmethyl, 4-(2xe2x80x2-methoxy)biphenylmethyl, 4-(4xe2x80x2-fluoro)biphenylmethyl, 4-(2xe2x80x2-trifluoromethanesulfonyl)biphenylmethyl, 4-(2xe2x80x2-cyclopropoxy)biphenylmethyl, 4-(2xe2x80x2,6xe2x80x2-dimethoxy)biphenylmethyl, 4-(2xe2x80x2,6xe2x80x2-diethoxy)biphenylmethyl, 4-(2xe2x80x2-benzyloxy)biphenylmethyl, 4-(2xe2x80x2-n-propoxy)biphenylmethyl, 4-(2xe2x80x2-cyano)biphenylmethyl, 4-(2xe2x80x2-n-propoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2-cyclopropylmethoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2-cyclobutylmethoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2-cyclohexylmethoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2-ethoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2-n-butoxy-6xe2x80x2-methoxy)biphenylmethyl, 4-(2xe2x80x2,6xe2x80x2-dipropoxy)biphenylmethyl, 4-(2xe2x80x2,6xe2x80x2-bis(cycloproxy))biphenylmethyl, 4-(2xe2x80x2-methoxy-5xe2x80x2-chloro)biphenylmethyl, 4-(2xe2x80x2-methoxy-3xe2x80x2-fluoro)biphenylmethyl, 4-(2xe2x80x2-fluoro-3xe2x80x2-methoxy)biphenylmethyl, 4-(2-thiazolyl)phenylmethyl, 4-(2,6-dimethoxy-4-pyrrolidinylmethylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-piperazinylmethylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-(4-methylpiperazinyl)methylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-(1-triazolyl)methylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-morpholinylmethylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-aminomethylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-(piperazinylcarbonyloxymethyl)phenyl)phenylmethyl, 4-(2,6-dimethoxy-4-(pyrrolidinylcarbonyloxymethyl)phenyl)phenylmethyl, 4-(2,6-dimethoxy-4-(dimethylaminoqarbonyloxymethyl)phenyl)phenylmethyl, 4-(2,6-dimethoxy-4-hydroxylmethylphenyl)phenylmethyl, 4-(2,6-dimethoxy-4-methoxymethylphenyl)phenylmethyl.
In one preferred subset of compounds of formula I are compounds of formula Ia: 
or a pharmaceutically acceptable salt thereof, wherein
A is a 4- to 8-membered monocyclic saturated heterocyclic ring having one to two heteroatoms chosen from O, S and S(O)m, optionally substituted with one to four substituents independently selected from oxo and Rb;
Y is
(1) a bond, or
(2) C1-5alkylene optionally substituted with one to four groups independently selected from Ra;
R1 is
(1) H,
(2) Cy optionally substituted with one to four substituents independently selected from Rb;
(3) OR5,
(4) OC(O)R5,
(4) OC(O)NRdRe,
(5) NRdRe,
(6) NRdC(O)R5,
(7) C(O)R5,
(8) C(O)OR5,
(9) C(O)NRdRe;
R3is
1) C1-10alkyl,
2) Ar1,
3) C1-10alkyl-Ar1,
4) Ar1xe2x80x94Ar2,
5) C1-10alkyl-Ar1xe2x80x94Ar2,
xe2x80x83wherein the alkyl group is optionally substituted with one to four substituents selected from Ra, and Ar1 and Ar2 are optionally substituted with one to four substituents independently selected from Rb. R1, R3, R5, Ra, Rb, Rd, Re, Ar1 and Ar2 are as defined under formula I.
In one preferred subset of formula Ia, A is tetrahydrofuranyl or tetrahydrothienyl each optionally substituted with one or two groups independently selected from C1-5alkyl, CH2OC(O)NRdRe, CH2NRdC(O)Re, CH2NRdSO2Re, CH2ORd, CH2xe2x80x94Ar1 (where Ar1 is optionally substituted with one to two groups selected from Ra), NRdRe, ORd and oxo, where Ra, Rd, Re and Ar1 are as defined under formula I. More preferably A is tetrahydrofuranyl.
In another preferred subset of formula Ia, Yxe2x80x94R1 is selected from hydrogen, C1-5alkyl, phenyl optionally substituted with one to three groups selected from Ra, C1-5alkylene-R1 (where R1 is hydroxy, C1-5alkoxy, C1-5alkanoyloxy, NRdRe, C(O)NRdRe, NRdC(O)C1-5alkyl, or phenyl optionally substituted with one to three groups selected from Rb), C(O)NRdRe, C(O)OR5, C(O)R5.
In another preferred subset of formula Ia, R3 is CH2xe2x80x94Ar1, wherein Ar1 is optionally substituted with one to three groups selected from Rb More preferably, R3 is benzyl optionally substituted with one to three groups selected from halogen, ORd, OC(O)NRdRe, NRdC(O)Re, C1-5alkyl optionally substituted with OC(O)NRdRe, C(O)OC1-5alkyl, hydroxy and C1-5alkoxy,.
In another preferred subset of compounds of formula Ia are compounds of formula Ib: 
or a pharmaceutically acceptable salt thereof, wherein A, Y, R1, Ar1 and Ar2 are as defined under formula Ia.
In one preferred subset of formula Ib, A is tetrahydrofuranyl or tetrahydrothienyl each optionally substituted with one or two groups independently selected from C1-5alkyl, CH2OC(O)NRdRe, CH2NRdC(O)Re, CH2NRdSO2Re, CH2ORd, CH2xe2x80x94Ar1 (where Ar1 is optionally substituted with one to two groups selected from Ra), NRdRe, ORd and oxo, where Ra, Rd, Re and Ar1 are as defined under formula I. More preferably A is tetrahydrofuranyl.
In another preferred subset of formula Ib, Yxe2x80x94R1 is selected from hydrogen, C1-5alkyl, phenyl optionally substituted with one to three groups selected from Ra, C1-5alkylene-R1 (where R1 is hydroxy, C1-5alkoxy, C1-5alkanoyloxy, NRdRe, C(O)NRdRe, NRdC(O)C1-5alkyl, or phenyl optionally substituted with one to three groups selected from Rb), C(O)NRdRe, C(O)OR5, C(O)R5.
In another preferred embodiment of formula Ib, Ar1 is phenyl. More preferably Ar2 is also phenyl, which is optionally substituted with one to three groups selected from Rb.
Examples of compounds of the present invention include:
xe2x80x9cAlkylxe2x80x9d, as well as other groups having the prefix xe2x80x9calkxe2x80x9d, such as alkoxy, alkanoyl, means carbon chains which may be linear or branched or combinations thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like.
xe2x80x9cAlkenylxe2x80x9d means carbon chains which contain at least one carbonxe2x80x94carbon double bond, and which may be linear or branched or combinations thereof. Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.
xe2x80x9cAlkynylxe2x80x9d means carbon chains which contain at least one carbonxe2x80x94carbon triple bond, and which may be linear or branched or combinations thereof. Examples of alkynyl include ethynyl, propargyl, 3-methyl-1-pentynyl, 2-heptynyl and the like.
Where an alkyl, alkenyl or alkynyl chain joins two other variables and is therefore bivalent, the terms alkylene, alkenylene and alkynylene are used.
xe2x80x9cCycloalkylxe2x80x9d means mono- or bicyclic saturated carbocyclic rings, each of which having from 3 to 10 carbon atoms. The term also includes monocyclic rings fused to an aryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.
xe2x80x9cArylxe2x80x9d means mono- or bicyclic aromatic rings containing only carbon atoms. The term, also includes aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of aryl include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like.
xe2x80x9cHeteroarylxe2x80x9d means a mono- or bicyclic aromatic ring containing at least one heteroatom selected from N, O and S, with each ring containing 5 to 6 atoms. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.
xe2x80x9cHeterocyclylxe2x80x9d means mono- or bicyclic saturated rings containing at least one heteroatom selected from N, S and O, each of said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen. The term also includes monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of xe2x80x9cheterocyclylxe2x80x9d include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2,3-dihydrofuro(2,3-b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, dihydroindolyl, and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4-pyridones attached through the nitrogen or N-substituted-(1H,3H)-pyrimidine-2,4-diones (N-substituted uracils).
xe2x80x9cHalogenxe2x80x9d includes fluorine, chlorine, bromine and iodine.
Optical Isomersxe2x80x94Diastereomersxe2x80x94Geometric Isomersxe2x80x94Tautomers
Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of Formula I.
Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.
Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed with compounds of Formula I.
Compounds of the Formula I may be separated into diastereoisomeric pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active acid as a resolving agent.
Alternatively, any enantiomer of a compound of the general Formula I or Ia may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.
Salts
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.
It will be understood that, as used herein, references to the compounds of Formula I are meant to also include the pharmaceutically acceptable salts.
Utilities
The ability of the compounds of Formula I to antagonize the actions of VLA-4 and/or xcex14xcex27 integrin makes them useful for preventing or reversing the symptoms, disorders or diseases induced by the binding of VLA-4 and or xcex14xcex2xcex27 to their various respective ligands. Thus, these antagonists will inhibit cell adhesion processes including cell activation, migration, proliferation and differentiation. Accordingly, another aspect of the present invention provides a method for the treatment (including prevention, alleviation, amelioration or suppression) of diseases or disorders or symptoms mediated by VLA-4 and/or xcex14xcex27 binding and cell adhesion and activation, which comprises administering to a mammal an effective amount of a compound of Formula I. Such diseases, disorders, conditions or symptoms are for example (1) multiple sclerosis, (2) asthma, (3) allergic rhinitis, (4) allergic conjunctivitis, (5) inflammatory lung diseases, (6) rheumatoid arthritis, (7) septic arthritis, (8) type I diabetes, (9) organ transplantation rejection, (10) restenosis, (11) autologous bone marrow transplantation, (12) inflammatory sequelae of viral infections, (13) myocarditis, (14) inflammatory bowel disease including ulcerative colitis and Crohn""s disease, (15) certain types of toxic and immune-based nephritis, (16) contact dermal hypersensitivity, (17) psoriasis, (18) tumor metastasis, (19) atherosclerosis, and (20) hepatitis.
Dose Ranges
The magnitude of prophylactic or therapeutic dose of a compound of Formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of Formula I and its route of administration. It will also vary according to the age, weight and response of the individual patient. In general, the daily dose range lie within the range of from about 0.001 mg to about 100 mg per kg body weight of a mammal, preferably 0.01 mg to about 50 mg per kg, and most preferably 0.1 to 10 mg per kg, in single or divided doses. On the other hand, it may be necessary to use dosages outside these limits in some cases.
For use where a composition for intravenous administration is employed, a suitable dosage range is from about 0.001 mg to about 25 mg (preferably from 0.01 mg to about 1 mg) of a compound of Formula I per kg of body weight per day and for cytoprotective use from about 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 1 mg to about 10 mg) of a compound of Formula I per kg of body weight per day.
In the case where an oral composition is employed, a suitable dosage range is, e.g. from about 0.01 mg to about 100 mg of a compound of Formula I per kg of body weight per day, preferably from about 0.1 mg to about 10 mg per kg and for cytoprotective use from 0.1 mg to about 100 mg (preferably from about 1 mg to about 100 mg and more preferably from about 10 mg to about 100 mg) of a compound of Formula I per kg of body weight per day.
For the treatment of diseases of the eye, ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of Formula I in an acceptable ophthalmic formulation may be used.
Pharmaceutical Compositions
Another aspect of the present invention provides pharmaceutical compositions which comprises a compound of Formula I and a pharmaceutically acceptable carrier. The term xe2x80x9ccompositionxe2x80x9d, as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of Formula I, additional active ingredient(s), and pharmaceutically acceptable excipients.
Any suitable route of administration may be employed for providing a mammal, especially a human with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The pharmaceutical compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic bases or acids and organic bases or acids.
The compositions include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (aerosol inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the conditions being treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulisers. The compounds may also be delivered as powders which may be formulated and the powder composition may be inhaled with the aid of an insufflation powder inhaler device. The preferred delivery systems for inhalation are metered dose inhalation (MDI) aerosol, which may be formulated as a suspension or solution of a compound of Formula I in suitable propellants, such as fluorocarbons or hydrocarbons and dry powder inhalation (DPI) aerosol, which may be formulated as a dry powder of a compound of Formula I with or without additional excipients.
Suitable topical formulations of a compound of formula I include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like.
In practical use, the compounds of Formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
In addition to the common dosage forms set out above, the compounds of Formula I may also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 3,630,200 and 4,008,719.
Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
The following are examples of representative pharmaceutical dosage forms for the compounds of Formula I:
Compounds of Formula I may be used in combination with other drugs that are used in the treatment/prevention/suppression or amelioration of the diseases or conditions for which compounds of Formula I are useful. Such other drugs may be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of Formula I. When a compound of Formula I is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of Formula I is preferred. Accordingly, the pharmaceutical compositions of the present invention include those that also contain one or more other active ingredients, in addition to a compound of Formula I. Examples of other active ingredients that may be combined with a compound of Formula I, either administered separately or in the same pharmaceutical compositions, include, but are not limited to: (a) other VLA-4 antagonists such as those described in U.S. Pat. No. 5,510,332, WO97/03094, WO97/02289, WO96/40781, WO96/22966, WO96/20216, WO96/01644, WO96/06108, WO95/15973 and WO96/31206; (b) steroids such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressants such as cyclosporin, tacrolimus, rapamycin and other FK-506 type immunosuppressants; (d) antihistamines (H1-histamine antagonists) such as bromopheniramine, chlorpheniramine, dexchlorpheniramine, triprolidine, clemastine, diphenhydramine, diphenylpyraline, tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine, cyproheptadine, antazoline, pheniramine pyrilamine, astemizole, terfenadine, loratadine, cetirizine, fexofenadine, descarboethoxyloratadine, and the like; (e) non-steroidal anti-asthmatics such as b2-agonists (terbutaline, metaproterenol, fenoterol, isoetharine, albuterol, bitolterol, salmeterol and pirbuterol), theophylline, cromolyn sodium, atropine, ipratropium bromide, leukotriene antagonists (zafirlukast, montelukast, pranlukast, iralukast, pobilukast, SKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005); (f) non-steroidal antiinflammatory agents (NSAIDs) such as propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone); (g) cyclooxygenase-2 (COX-2) inhibitors such as celecoxib; (h) inhibitors of phosphodiesterase type IV (PDE-IV); (i) antagonists of the chemokine receptors, especially CCR-1, CCR-2, and CCR-3; (j) cholesterol lowering agents such as HMG-CoA reductase inhibitors (lovastatin, simvastatin and pravastatin, fluvastatin, atorvastatin, and other statins), sequestrants (cholestyramine and colestipol), nicotinic acid, fenofibric acid derivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), and probucol; (k) anti-diabetic agents such as insulin, sulfonylureas, biguanides (metformin), a-glucosidase inhibitors (acarbose) and glitazones (troglitazone, pioglitazone, englitazone, MCC-555, BRL49653 and the like); (1) preparations of interferon beta (interferon beta-1a, interferon beta-1b); (m) anticholinergic agents such as muscarinic antagonists (ipratropium bromide); (n) other compounds such as 5-aminosalicylic acid and prodrugs thereof, antimetabolites such as azathioprine and 6-mercaptopurine, and cytotoxic cancer chemotherapeutic agents.
The weight ratio of the compound of the Formula I to the second active ingredient may be varied and will depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the Formula I is combined with an NSAID the weight ratio of the compound of the Formula I to the NSAID will generally range from about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. Combinations of a compound of the Formula I and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
Compounds of the present invention may be prepared by procedures illustrated in the accompanying schemes. In the first method (Scheme 1), a resin-based synthetic strategy is outlined where the resin employed is represented by the ball (∘). An N-Fmoc-protected amino acid derivative A (Fmoc=fluorenylmethoxycarbonyl) is loaded on to the appropriate hydroxyl-containing resin (the choice of resin being dependent on type of linker used, in this case Wang resin was utilized) using 1-ethyl-3-(3xe2x80x2-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt) in a solvent such as methylene chloride and teterahydrofuran or dimethylformamide (DMF) to give B. The Fmoc protecting group is removed with piperidine in DMF to yield free amine C. A carboxylic acid D is then coupled to the amine using a reagent such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in the presence of HOBt and diisopropyl ethyl amine or any of the other well known amide coupling reagents under appropriate conditions: EDC, DCC or BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate) to give E. The final product is removed from the resin with strong acid (in this instance, trifluoroacetic acid (TFA in the presence of 5% water) to yield compounds of the present invention F. 
In the second method (Scheme 2), standard solution phase synthetic methodology is outlined. Many amino acid derivatives are commercially available as the t-butyl or methyl esters and may be used directly in the synthesis outlined below. Amino acid t-butyl esters B may be prepared from amino acids C directly by treatment with isobutylene and sulfuric acid in diglyme or dioxane. Alternatively, N-Boc-protected amino acid derivative A (Boc=tert-butyloxycarbonyl) is treated with tert-butyl 2,2,2-trichloroacetimidate in the presence of boron trifluoride etherate followed by treatment with strong acid (HCl in ethyl acetate or sulfuric acid in t-butyl acetate) to remove the t-BOC group to yield tert-butyl ester B which is subsequently coupled to carboxylic acid D in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), HOBt, and diisopropylethylamine (DIEA) in methylene chloride to yield amide E. The ester is then hydrolysed (in the case of t-butyl ester with 50% TFA in methylene chloride and for the methyl ester by treatment with 1N sodium hydroxide solution in methanol or dioxane) to provide compounds of the present invention F. 
In a third method (Scheme 3), a late stage intermediate aryl bromide or iodide is coupled to an appropriately substituted aryl or heteroaryl boronic acid to give a subset of compounds of the present invention (R3=biaryl-substituted-alkyl or heteroaryl-aryl-substituted-alkyl, R2=hydrogen). For example, 4-iodo or 4-bromophenyl-derivative A is converted to the t-butyl ester B by treatment with isobutylene and sulfuric acid. Alternatively the N-Boc-4-iodo- or 4-bromo-phenyl-derivative C is reacted with tert-butyl 2,2,2-trichloroacetimidate in the presence of boron trifluoride etherate in methylene chloride-cyclohexane followed by treatment with strong acid (HCl in ethyl acetate or sulfuric acid in t-butyl acetate) to remove the t-BOC group to yield tert-butyl ester B which is subsequently coupled with C in the presence of (for example) EDC, HOBt and NMM to yield amide E. Substituted aryl or heteroaryl boronic acids are coupled to E in the presence of a palladium(0) reagent, such as tetrakis(triphenylphosphine)palladium under Suzuki conditions (N. Miyaura et al., Synth. Commun., 1981, 11, 513-519), followed by removal of the tert-butyl ester using a strong acid (TFA) to yield the desired product F. If the aryl or heteroaryl boronic acid is not commercially available, but the corresponding bromide or iodide is, then the bromide or iodide can be converted into the desired boronic acid by treatment with an alkyllithium reagent in tetrahydrofuran at low temperature followed by addition of trimethyl or triisopropyl borate. Hydrolysis to the boronic acid can be effected by treatment of the intermediate with aqueous base and then acid. 
Alternatively, the aryl coupling reaction may be performed by application of Stille-type carbonxe2x80x94carbon bond forming conditions (Scheme 4). (A. M. Echavarren and J. K. Stille, J. Am. Chem. Soc. 1987, 109, 5478-5486). The aryl bromide or iodide intermediate A is converted into its trialkyltin derivative B using hexamethylditin in the presence of a palladium(0) catalyst and lithium chloride and then reacted with an appropriately substituted aryl or heteroaryl bromide, iodide, or triflate in the presence of a palladium reagent, such as tetrakis(triphenylphosphine)-palladium(0) or tris(dibenzylideneacetone)dipalladium(0), in a suitable solvent, such as toluene, dioxane, DMF, or 1-methyl-2-pyrrolidinone, followed by the removal of the tert-butyl ester using strong acid (TFA) to yield the desired product C. Biphenyl amino acids suitable for attachment to resin (C where R1 is fluorenylmethyloxy) may be prepared by this route as well. Superior coupling conversions and rates may be elicited by application of the method of Farina (J. Org. Chem. 5434, 1993) 
Compounds wherein the middle ring is heteroaryl (G) may be prepared (Scheme 5) in a similar fashion starting from the appropriate heteroaryl bromide or iodide C using Suzuki-type conditions as depicted in Scheme 3 or from the corresponding heteroaryl trimethyltin using Stille-type conditions as depicted in Scheme 4. The requisite heteroaryl halides C may be prepared via conventional electrophilic halogenation of the N-Boc-heteroaryl-alanine tert-butyl ester intermediate B. B may be prepared from the known aliphatic iodo intermediate A in carbonxe2x80x94carbon bond formation using zinc/copper couple and palladium(II) (M. J. Dunn et al., SYNLETT 1993, 499-500). 
Step A. N-FMOC-(L)-4-Iodophenylalanine, t-Butyl Ester.
To a solution of 15 g (51 mmol) of (L)4-iodo-phenylalanine in 100 ml of diglyme and 15 ml of concentrated H2SO4 was added 30 ml of condensed isobutylene. The vessel was agitated overnight and the crude product was diluted with 100 ml of ethyl acetate. The solution was added to excess sodium hydroxide solution while maintaining the temperature below 30xc2x0 C. A white precipitate formed which dissolved upon addition of sodium hydroxide solution. The resulting mixture was filtered and the aqueous phase was extracted with ethyl acetate. The combined extracts were washed with brine and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give a solution of the product in diglyme. The solution was diluted with 200 ml of ether and was treated with excess 1N HCl in ether with rapid stirring. The resulting precipitate was collected and dried in vacuo after washing with ether. A white solid (9.01 g) was collected of 4-iodo-phenylalanine t-butyl ester hydrochloride. To a suspension of 5.1 g (13.3 mmol) of the amine hydrochloride in 30 ml of methylene chloride was added 3.6 g (27 mmol) of diisopropyl ethyl amine followed by 3.43 g (0.013 g) of FMOCCl. The solution was stirred overnight at room temperature, washed with 1N HCl solution (3xc3x9750 ml), water (1xc3x9750 ml), saturated sodium carbonate solution (2xc3x9750 ml) and brine (1xc3x9750 ml). The solution was dried over MgSO4, filtered and concentrated in vacuo to give 6.43 g of N-FMOC-(L)-4-iodophenylalanine, t-butyl ester as a white foam.
300 MHz 1H NMR (CDCl3): d 1.44 (s, 9 H); 3.05 (d, 2H);4.20-4.60 (m, 4 H); 5.30 (m, 1H); 6.90 (d, 2H), 7.30-7.80 (m, 12H).
Step B. N-FMOC-(L)-4-Trimethylstannyl-phenylalanine, tert-Butyl Ester.
In a dry 250 ml round bottom flask was added 6.20g (10.5 mmol) of the product of Step A, 0.48 g (115 mmol) LiCl and 0.6 g (0.52 mmol) of palladium tetrakistriphenylphosphine followed by 50 ml of dry dioxane. The mixture was stirred for 5 minutes. 5.2 g (15.8 mmol) of hexamethylditin was added and the reaction mixture was degassed and then heated at 90xc2x0 C. The reaction mixture gave a black suspension after 15 minutes. Completion of the conversion was determined by TLC (10% EtOAc/hexanes; sm r.f.=0.3, product r.f.=0.4). The mixture was diluted with 100 ml of hexanes and stirred to give a precipitate. The suspension was filtered through celite and concentrated in vacuo to give a gum. The residue was purified by flash chromatography over silica gel eluting with 10% EtOAc/hexanes to give 5.02 g of the stannane (77% yield).
300 MHz 1H NMR (CDCl3): d 0.30 (s, 9 H); 1.45 (s, 9H); 3.20 (d, 2H), 4.20-4.60 (m, 4H); 5.29 (d, 1H); 7.12 (d, 2H); 7.22-7.45 (m, 6H); 7.59 (d, 2H), 7.75 (d, 2H).
Step C. N-FMOC-(L)-4-(2xe2x80x2-Cyanophenyl)phenylalanine, tert-Butyl Ester.
In a clean, dry round bottom flask fitted with a reflux condenser vented through a three way valve attached to a vacuum source and nitrogen gas was added 1.56 g (6.8 mmol) of 2-iodobenzonitrile, 0.117 (0.12 mmol) of tris(dibenzylidineacetone)-dipalladium (0), 0.8 g (19 mmol) of LiCl and 0.15 g (0.5 mmol) of triphenylarsine followed by 30 ml of N-methylpyrrolidinone (NMP). The mixture was degassed and stirred for 10 minutes at which time most of the catalyst mixture had dissolved. 3.9 g (6.21 mmol) of the product of Step B was added in 10 ml of NMP and the reaction was heated to 80xc2x0 C. for 90 minutes. TLC (10% EtOAc/hexanes) indicated complete consumption of stannane (rf=0.4) and formation of the desired product (rf=0.1). The solution was cooled to room temperature and diluted with 50 ml of EtOAc. The solution was stirred with 20 ml of saturated KF for 20 minutes. The mixture was diluted with 200 ml of EtOAc and washed with water (6xc3x9775 ml), brine (1xc3x9750 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo and the residue was purified by Biotage Flash chromatography over silica gel eluting with 20% EtOAc/hexanes to give 1.91 g (54% yield) of the title compound.
300 MHz 1H NMR (CDCl3): d 1.45 (s, 9H); 3.19 (d, 2H); 4.20-4.68 (m, 4H); 5.40 (d, 1H); 7.25-7.55 (m, 12H); 7.65 (m, 2H), 7.80 (d, 2H).
Step D. N-FMOC-(L)-4-(2xe2x80x2-Cyanophenyl)phenylalanine.
2.4 g of the product of Step C was treated with 50 ml of a mixture of 50% trifluoroacetic acid in methylene chloride. The reaction mixture was concentrated in vacuo. The residue was azeotropically dried by concentration from toluene to give the desired product as a foam.
300 MHz 1H NMR (CD3OD): d 3.02 (dd, 1H); 3.30 (dd, 1H); 4.05-4.35 (m, 3H); 4.52 (m, 1H); 7.10-7.50 (m, 12H); 7.60 (m, 2H), 7.78 (d, 2H).
Step A. N-(Boc)-(L)4-Iodo-phenylalanine, tert-Butyl Ester.
To a suspension of 7.5 g (0.019 m) of 4-iodophenylalanine t-butyl ester (Reference Example 1 Step A prior to treatment with HCl) in 100 ml of dichloromethane was added 2.52 g 0.019 m of diisopropyl ethyl amine followed by 4.14 g of ditertbutyldicarbonate. The reaction mixture was stirred overnight at room temperature, washed with 1N HCl (2xc3x9725 ml), water (2xc3x9725 ml), saturated NaHCO3 (1xc3x9725 ml), brine (1xc3x9725 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo to give the desired product as a gum 8.8 g (100% yield).
300 MHz 1H NMR (CDCl3): xcex4 1.39 (s, 18H); 2.98 (AB, 2H); 4.4 (dd, 2H); 5.0 bd, 1H); 6.92 (d, 2H); 7.62 (d, 2H).
Step B. N-(Boc)-(L)-4-(2xe2x80x2-Methoxyphenyl)phenylalanine, tert-Butyl Ester.
7.97 g (0.018 m) of the product of Step A was dissolved in 160 ml of 2:1 toluene:ethanol. To this solution was added 2.99 g (0.0198 m) 2-methoxyphenylboronic acid, 0.69 g of tetrakistriphenylphosphine palladium (0) and 22.7 ml (0.45 m) of 2.0 M sodium carbonate in water. The reaction mixture was degassed three times and then heated at 90xc2x0 C. for 90 minutes at which time the reaction mixture was black. The mixture was diluted with 300 ml of ethyl acetate, washed with water (3xc3x97150 ml) and brine (2xc3x97100 ml), and dried over MgSO4. The mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography over silica gel eluting with 10% EtOAc/hexanes to give 6.89 g (88% yield) of the desired product as a white solid.
300 MHz 1H NMR (CDCl3): xcex4 1.45 (s, 18H); 3.10 (d, 2H); 3.80 (s, 3H); 4.5 (dd, 2H); 5.1 bd, 1H); 7.0 (m, 2H); 7.22 (d, 2H); 7.30 (d, 2H); 7.49 (d, 2H); 7.62 (d, 2H).
Step C. N-(FMOC)-(L)4-(2xe2x80x2-Methoxyphenyl)phenylalanine.
To a solution of 4.85 g (0.0113 m) of the product of Step B in 100 ml of t-butyl acetate was added 5.53 g (0.056 m) of concentrated sulfuric acid. The solution was stirred at room temperature for 2 hours and then carefully neutralized by addition of saturated aqueous NaHCO3 solution. The solution was washed with NaHCO3 solution, dried over NaSO4, filtered and concentrated in vacuo. To a solution of 4.42 g of amine in 150 ml of methylene chloride was added at 0xc2x0 C. 1.74 g (13.5 mmol) of diisopropylethyl amine followed by 3.48 g (13.5 mmol) of FMOCCl. The solution was stirred for 2 hours and washed with 1N HCl (3xc3x9750 ml), saturated NaHCO3 solution (2xc3x9750 ml) and brine (1xc3x9750 ml). The mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography over silica gel eluting with a gradient of 10-25% EtOAc/hexanes to give 7.10 g (88% yield) of the desired product as a glass. The material was dissolved in 125 ml of 50% trifluoracetic acid/methylene chloride and stirred at room temperature for 2.5 hours. The solution was concentrated in vacuo and the residue was redissolved in toluene and concentrated in vacuo to give 7.01 g of the desired product. 96% pure by HPLC (254 nm).
300 MHz 1H NMR (CDCl3): xcex4 3.20 (m, 2H); 3.76 (s, 3H); 4.21 (t, 1H); 4.41 (m, 4H); 4.76 (dd, 1H); 5.32 (d, 1H); 6.8-7.8 (m, 16H).
Step A. N-(Boc)-(L)-Tyrosine tert-Butyl Ester.
To a solution of 9.82 g (0.041 m) of (L)-tyrosine, tert-butyl ester in 150 ml of methylene chloride and 20 ml of DMF was added 5.2 g (0.04 m) of triethyl amine followed by 9.03 g (0.04 m) of ditertbutyldicarbonate. The reaction mixture was stirred for 2 hours at room temperature and was then washed with 1 N HCl (3xc3x9750 ml), NaHCO3 solution (1xc3x9750 ml) and brine (1xc3x9750 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo to give 13.59 g (98% yield) of a white solid.
300 MHz 1H NMR (CDCl3): 1.42 (s, 18H); 2.95 (d, 2H); 4.39 (dd, 1H); 5.01 (d, 1H); 6.15 (s, 1H); 6.70 (d, 2H); 7.00 d, 2H).
Step B. N-(Boc)-(L)-4-(1-Pyrrolidino-carbonyloxy)phenylalanine, tert-Butyl Ester.
To a solution of 8.18 g (0.024 m) of the product of Step A in a clean, dry flask dissolved in 100 ml of THF under a dry nitrogen atmosphere was added at 0xc2x0 C. 25.5 ml (0.025 m) of a 1M solution of sodium hexamethyldisilazide in THF. The solution was stirred for 20 minutes. A solution of 3.2 g (0.024 m) of pyrrolidine carbamoyl chloride in 10 ml of THF was added. The reaction mixture was allowed to warm to room temperature and was stirred for 48 hours. The solution was diluted with 100 ml of ethyl acetate and was washed with 1N HCl (3xc3x9775 ml), saturated NaHCO3 (1xc3x9775 ml), 1N NaOH (2xc3x9775 ml) and brine (1xc3x9775 ml) and was dried over MgSO4. The mixture was filtered and concentrated in vacuo and the residue was recrystalized from ethyl acetate/hexanes to give 8.6 g of a white solid.
300 MHz 1H NMR (CDCl3): xcex4 1.40 (s, 9H); 1.41 (s, 9H); 1.92 (m, 4H); 3.02 (d, 2H); 3.45 (t, 2H); 3.55 (t, 2H); 4.42 (dd, 1H); 4.99 (d, 1H); 7.05 (d, 211); 7.15 (d, 2H).
Step C. N-(FMOC)-(L)-4-(1-Pyrrolidino-carbonyloxy)phenylalanine.
The method of Reference Example 2 Step C was applied to 8.1 g (0.018 m) of the product of Step B to give 6.27 g of the title compound as a foam. 71% overall yield.
300 MHz 1H NMR (CDCl3): xcex4 1.97 (bs, 4H); 3.12 (bd, 2H); 3.4-3.6 (2 bm, 4H); 4.20 (m, 1H); 4.30-4.50 (m, 2H); 4.69 m, 1H); 5.59 (t, 1H); 7.00-7.42 (m, 8H); 7.55 (bm, 2H); 7.77 (d, 2H).
To a solution of 4.85 g (0.0113 m) of the product of Reference Example 2 Step B in 100 ml of t-butyl acetate was added 5.53 g (0.056 m) of concentrated sulfuric acid. The solution was stirred at room temperature for 2 hours and then carefully neutralised by addition of saturated aqueous NaHCO3 solution. The solution was washed with NaHCO3 solution, dried over NaSO4, filtered and concentrated in vacuo. The residue was dissolved in 50 ml of ether and treated with anhydrous HCl gas with stirring to give a white precipitate. The solid was collected by filtration, washed with ether and dried in vacuo to give the desired product. 300 MHz
1H NMR (CD3OD): 1.45 (s, 9H); 3.20 (d, 2H); 3.79 (s, 3H); 4.21 (t, 1H); 7.03 (m, 2H); 7.28 (m, 2H); 7.31 (d, 2H); 7.50 (d, 2H).
To a solution of N,N-diisopropylamine (15 ml, 106 mmol) in THF (40 ml) at 0xc2x0 C. was added n-BuLi (99 mmol, 1.6 M/Hex). After stirring the mixture for 30 min. DMPU (12 ml, 99 mmol) and tetrahydro-2-furoic acid (4.2 ml, 44 mmol) were added at 0xc2x0 C. After 1 hr at 0xc2x0 C., iodomethane (5.4 ml, 88 mmol) was added and the reaction mixture was allowed to warm to room temperature. After stirring overnight, the reaction mixture was partitioned between ethyl acetate and 1N HCl. The aqueous layer was extracted with ethyl acetate (2xc3x97500 ml) and chloroform (500 ml). The extracts were washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography with methylene chloride/methanol/acetic acid=97:3:0.5 to afford the desired product as an oil (2.5 g).
400 MHz 1H NMR (CDCl3) xcex4 3.99 (m, 2H); 2.38 (m, 1H); 1.95 (m, 3H); 1.02 (s, 3H)
2-Benzyl-2-tetrahydrofuroic acid, 2-phenylethyl-2-tetrahydrofuroic acid, 2-methyl-2-tetrahydropyranoic acid, 2-benzyl-2-tetrahydropyranoic acid and 2-phenylethyl-2-tetrahydropyranoic acid were prepared by the alkylation procedures described for 2-methyl-2-tetrahydrofuroic acid in Reference Example 5 using commercially available alkyl- or aralkyl-halides and the corresponding carboxylic acid.
To a vigorously stirred solution of tetrahydropyran-2-methanol (2.32 g, 40 mmol) in a mixture of CH3CN (20 ml), CCl4 (20 ml) and water (10 ml) at 0xc2x0 C. was added NaIO4 (9.42 g, 44 mmol) and then RuCl3H2O(414 mg, 2 mmol). After 20 min, the cooling bath was removed. After 40 min at room temperature, TLC showed reaction was complete. The reaction mixture was partitioned into ethyl acetate and ice-cold Na2S2O5 solution. More Na2S2O5 was added until all the brown material dissolved and a greenish mixture was obtained. The aqueous portion was extracted with more ethyl acetate and combined layer was washed with 10% Na2S2O5, H2O, brine, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography using methylene chloride/methanol/acetic acid=95:5:0.5 to afford the product as an oil (1.5 g).
400 MHz 1H NMR (CDCl3): xcex4 4.12 (m, 1H); 3.98 (m, 1H); 3.56 (m, 1H); 2.09 (m, 1H); 1.94 (m, 1H); 1.58 (m, 4H).
Step A. 2-Hydroxyl-2-phenyl-4-pentenoic Acid, Ethyl Ester.
The title compound was prepared according to a literature procedure (T. Akiyama, K. Ishikawa and S. Ozaki, Chemistry letters 1994, 627). To a solution of ethyl benzoylformate (2.5 g, 14.0 mmol) and allyl trimethylsilane (2.7 ml, 16.9 mmol) in 31 ml of methylene chloride (anhydrous) at xe2x88x9278xc2x0 C. was added 1.6 ml of tin chloride (14.28 mmol) dropwise. The cooling bath was removed after the addition was complete. After the reaction mixture was stirred at room temperature for 5 min., TLC showed the reaction was complete. Triethylamine (3.5 ml) and water (150 ml) were added to quench the reaction. Extraction with ethyl acetate was complicated by the emulsion which was broken up by filtration through celite. The extracts were washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography with methylene chloride/hexane=2.5:97.5 to afford 2-hydroxyl-2-phenyl-4-pentenoic acid, ethyl ester as an oil (1.2 g). 400 MHz 1H NMR (CDCl3): xcex4 7.59 (m, 2H); 7.29 (m, 3H); 5.79 (m, 1H); 5.13 (m, 2H); 4.23 (m, 2H); 2.96 (m, 1H); 2.74 (m, 1H); 1.26 (t, J=7.0 Hz, 3H).
Step B. 4-Isopropoxy-2-phenyl-2-tetrahydrofuroic Acid, Ethyl Ester.
To a 10 ml round bottom flask were placed PdNO2Cl(CH3CN)2 (20 mg, 0.077 mmol) and CuCl2 (20 mg, 0.308 mmol) and isopropyl alcohol (2.5 ml). The reaction vessel was purged with oxygen, fitted with an oxygen balloon and heated to 55xc2x0 C. for 2 h. After cooling to 30xc2x0 C., the homoallyl alcohol from Step A (356 mg, 1.54 mmol) was added in 5 ml of isopropyl alcohol and stirred overnight at 30xc2x0 C. The catalyst was removed by filtration through Al2O3. The filtrate was concentrated and purified by preparative TLC using ethyl acetate/hexane=5:95 to give ethyl-4-isopropoxy-2-phenyl-2-tetrahydrofuroate as an oil (300 mg).
400 MHz 1H NMR (CDCl3): xcex4 4.13 (m, 4H); 3.02 (m, 1H); 2.12 (m, 1H); 1.91 (m, 2H); 1.18 (m, 9H).
Step C. 2-Phenyl-2-tetrahydrofuroic Acid, Ethyl Ester.
To a solution of 4-isopropoxy-2-phenyl-2-tetrahydrofuroic acid, ethyl ester (189 mg, 0.68 mmol) in 4 ml of TFA was added triethylsilane (395 mg, 3.40 mmol). After heating at 70xc2x0 C. for 4 h, the reaction mixture was poured into NaHCO3 (saturated) and extracted with ethyl acetate. The extracts were washed with brine and dried over MgSO4, filtered, and concentrated. The residue was purified by preparative TLC eluting with ethyl acetate/hexane=5:95 to give ethyl 2-phenyl-2-tetrahydrofuroate (170 mg).
400 Mhz 1H NMR (CDCl3): xcex4 4.13 (q, J=7.5 Hz, 2H); 4.83 (m, 2H); 2.81 (m, 1H); 1.94 (m, 2H); 1.19 (t, J=7.5 Hz, 3H).
Step D. 2-Phenyl-2-tetrahydrofuroic Acid
To a solution of ethyl 2-phenyl-2-tetrahydrofuroate (170 mg, 0.77 mmol) in methanol (6 ml) was added 0.5N NaOH (1.5 ml, 0.75 mmol). After stirring at room temperature overnight, it was partitioned between dilute acetic acid and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic layer was washed with brine and dried over MgSO4, filtered and concentrated to afford the title compound as a white crystalline solid which was used without further purification. 400 MHz 1H NMR (CD3OD): xcex4 7.54 (m, 2H); 7.34 (m, 3H); 4.07 (m, 2H); 2.79 (m, 1H); 2.18 (m, 1H); 1.98 (m, 2H).
Step A. 4-Fluorobenzoyl Cyanide
This compound was prepared according to a literature procedure (M. M. Kayser et al., J. Labelled Compd. Radiopharm. 1987, 25, 301). CuCN (6 g, 67 mmol) and 4-fluorobenzoylchloride (8 ml, 67 mmol) were added to a sealed tube which was placed in an oil bath pre-heated to 140xc2x0 C. The temperature was increased to 225xc2x0 C. After 5 h, it was cooled, diluted with ethyl ether and filtered through a pad of celite. The filtrate was concentrated under reduced pressure and pumped to give a dark brown oil which was used in the subsequent hydrolysis step without further purification.
Step B. 4-Fluorophenyl Glyoxylic Acid
In a 250 ml round bottom flask were placed the crude 4-fluorobenzoyl cyanide (9 g, 67 mmol) and conc. HCl (46 ml). The mixture was stirred at room temperature. for 5 days. The resulting clear yellow solution was poured into 400 ml of ice water and extracted with ethyl acetate. The combined extracts were washed with water, dried over MgSO4, concentrated under reduced pressure and pumped to give a yellow solid (8 g) which was used in the subsequent esterification step without further purification.
Step C. 4xe2x80x2-Fluorophenyl Glyoxylic Acid, Methyl Ester.
To a solution of 4-fluorophenyl glyoxylic acid (100 mg, 0.595 mmol) in ethyl ether (5 ml) was added trimethylsilyl diazomethane (2 M/hexane, 1 ml) at 0xc2x0 C. TLC showed reaction was complete after 1 h. The ether was evaporated to leave a yellow oil. Chromatography with hexane/ether (95:5) gave methyl 4-fluorophenylglyoxalate as a pale yellow oil (75 mg). 500 MHz 13C NMR (CDCl3): xcex4 184.11; 167.85; 165.79; 163.59; 133.04; 132.96; 128.97; 116.33; 116.16; 115.90; 115.72; 52.85.
Step D. 2-(4xe2x80x2-Fluorophenyl)-3-trimethylsilyl-2-tetrahydrofuroic Acid, Methyl Ester.
To a solution of allyl trimethyl silane (0.04 ml, 0.245 mmol) and methyl 4xe2x80x2-fluorophenylglyoxalete (40 mg, 0.204 mmol) in 1.5 ml of methylene chloride at xe2x88x9278xc2x0 C. was added SnCl4 (0.224 ml, 0.224 mmol). The cooling bath was removed after the addition was complete. After 5 min at room temperature., the reaction was quenched by addition of triethylamine and water and extracted with ethyl acetate. The combined extracts were washed with brine, dried over MgSO4, filtered and concentrated. The residue was purified by preparative TLC eluting with hexane/ethyl acetate=95:5 to give methyl-2-(4-fluorophenyl)-3-trimethylsilyl-2-tetrahydrofuroate (16 mg).
300 MHz 1H NMR (CDCl3): xcex4 7.51 (m, 2H); 7.03 (m, 2H); 4.26 (t, 1H); 3.83 (dd, 1H); 3.72 (s, 3H); 2.38 (m, 2H); 1.31 (m, 1H); 0.00680 (s, 9H).
Step E. 2-(4-fluorophenyl)-2-tetrahydrofuroic Acid
To a solution of methyl-2-(4-fluorophenyl)-3-trimethylsilyl-2-tetrahydrofuroate (16 mg, 0.0541 mmol) in DMSO (0.5 ml) was added potassium t-butoxide (30 mg, 0.270 mmol). The resulting mixture was heated at 60xc2x0 C. overnight. TLC showed reaction was complete. The reaction mixture was poured into HCl (1N) and extracted with ethyl acetate. The combined organic layer was dried over MgSO4 and concentrated. The residue was purified by preparative TLC eluting with methylene chloride/methanol/acetic acid=95:5:0.5 to give 2-(4-fluorophenyl)-2-tetrahydrofuroic acid (14 mg).
300 MHz 1H NMR (CDCl3): xcex4 7.56 (m, 2H); 7.05 (m, 2H); 4.19 (m, 1H); 4.11 (m, 1H); 2.82 (m, 1H); 2.23 (m, 1H); 2.01 (m, 2H).
Step A. Thiophene-2-glyoxylic, Ethyl Ester.
Thiophene-2-glyoxylic acid (2 g, 12.8 mmol) was added to anhydrous ethanol (100 ml) which was saturated with HCl by bubbling HCl gas for 2 min. The resulting mixture was stirred at room temperature. overnight. TLC showed the reaction was complete. Solvent was removed and the residue was purified by chromatography over silica gel eluting with ethyl acetate/hexane 10:90 to give ethyl thiophene-2-glyoxylate (1.5 g).
Step B. 2-Hydroxy-2-(2-thienyl)-5-(tetrahydro-2H-pyranyloxy)-pentanoic Acid, Ethyl Ester.
Sec-BuLi (1.7 ml, 2.17 mmol, 1.3 M/cyclohexane) was added to a solution of 2-(3-bromopropoxy)tetrahydro-2H-pyran (254 mg, 1.09 mmol) in THF (6 ml) cooled to xe2x88x9250xc2x0 C. After 30 min at xe2x88x9250xc2x0 C., the reaction mixture was cooled to xe2x88x9278xc2x0 C. and then transferred to a solution of ethyl thiophene-2-glyoxylate (200 mg, 1.09 mmol) in THF (6 ml) at xe2x88x9278xc2x0 C. After stirring at xe2x88x9278xc2x0 C. for 30 min, TLC showed reaction was complete. The reaction mixture was poured into NH4Cl (saturated) and extracted with ethyl acetate. The extract was washed with water, brine, dried over MgSO4 and concentrated. The residue was purified by silica gel chromatography using ethyl acetate/hexane 10:90 to afford the product as an oil (38 mg).
300 MHz 1H NMR (CDCl3): xcex4 7.22 (dd, 1H); 7.18 (dd, 1H); 6.96 (dd, 1H); 4.57 (m, 1H); 4.28 (m, 2H); 3.79 (m, 2H); 3.43 (m, 2H); 2.19 (m, 2H); 1.85-1.52 (m, 8H); (t, 3H).
Step C. 2-(2xe2x80x2-Thienyl)-2-tetrahydrofuroic Acid, Ethyl Ester.
To a solution of 2-hydroxy-2-(2-thienyl)-5-(tetrahydro-2xe2x80x2H-pyranyloxy)-pentanoic acid, ethyl ester (38 mg, 0.116 mmol) in methylene chloride (1.5 ml) at xe2x88x9220xc2x0 C. was added 2,6-lutidine and trimethylsilyl trifluoromethanesulfonate (0.023 ml, 0.128 mmol). The resulting solution was stirred at this temperature for 1 h. TLC showed reaction was incomplete. One more equiv of the reagents was added. TLC showed the reaction was complete after it was stirred for an additional 1 h. The reaction mixture was poured into HCl (0.5 N) and extracted with ethyl acetate. The extract was dried over MgSO4, filtered and concentrated. The residue was purified by preparative TLC eluting with ethyl acetate/hexane 10:90 to 2-(2xe2x80x2-thienyl)-2-tetrahydrofuroic acid, ethyl ester (5 mg).
300 MHz 1H NMR (CDCl3): xcex4 7.24 (dd, 1H); 7.08 (dd, 1H); 7.08 (dd, 1H); 6.96 (dd, 1H); 4.21 (q, 2H); 4.10 (m, 1H); 2.70 (m, 1H); 2.34 (m, 1H); 2.01 (m, 2H); 126 (t, 3H).
Step D. 2-(2xe2x80x2-thienyl)-2-tetrahydrofuroic Acid
To a solution of 2-(2xe2x80x2-thienyl)-2-tetrahydrofuroic acid, ethyl ester (5 mg, 0.0221 mmol) in methanol (0.5 ml) was added NaOH (0.066 ml, 0.0332 mmol, 0.5 N). After it was stirred at r. t. overnight, it was poured into water and acidified by adding glacial acedic acid dropwise until pH 4-5 was reached. The aqueous portion was extracted with ethyl acetate (2xc3x9750 ml). The extracts were dried over MgSO4, filtered, and concentrated to give (2xe2x80x2-thienyl)-2-tetrahydrofuroic acid (3.5 mg) which was used in the subsequent reaction without further purification.
300 MHz 1H NMR (CDCl3): xcex4 7.51 (m, 2H); 7.03 (m, 2H); 4.26 (t, 1H).
To a solution of racemic 2-methyl-2-tetrahydrofuranoic acid (50 g, 0.384 mole) in 75 mL of ethanol at xe2x88x9260xc2x0 C. was added (S)-(xe2x88x92)-xcex1-methylbenzylamine (0.384 mole, 50.5 mL of 98% pure material) dropwise via an addition funnel. Upon completion of the addition, the reaction mixture was stirred at xe2x88x9260xc2x0 C. for another 30 minutes before 75 mL of cold acetone (xe2x88x9260xc2x0 C.) was added. The precipitant was filtered over sintered glass (pore C) and the solids were washed with cold acetone (xe2x88x9260xc2x0 C.). Some solid formed in the filtrate during the process. This was collected over sintered glass and washed as well with cold acetone. This process was repeated until no further material precipitated. Altogether, 88 g of a slightly reddish brown and tacky solid was obtained, wet with ethanol. All the funnels and adapters were carefully washed with methanol and the material collected, pumped down to dryness, redissolved in ethanol and added to the filtrate. Solvent was removed from this mixture until salt began to form. The salt was collected over sintered glass exhaustively as described above for the first batch. This provided 13 g more of the acid-amine salt.
The major batch obtained above (88 g) was dissolved in 240 mL hot (60-65xc2x0 C.) ethanol and cooled to room temperature gradually. After 24 h, no crystals had formed. Crystallization was induced by gradual and careful cooling to 8xc2x0 C. when fine needles began to form. This mixture, after filtration over sintered glass and washing with cold acetone (xe2x88x9260xc2x0 C.), provided 10 g of a free-flowing salt. The filtrate obtained was put on the rotovap until crystal began to form in the flask. It was then allowed to crystallize at 6xc2x0 C. This provided 3 g more of the salt. The filtrate was again placed on the rotovap but more ethanol was removed (so that more crystals were formed in the flask) before recrystallizing the mixture at 4-5xc2x0 C. This was repeated and together obtained 30 g more of the salt after filtration and washing as described above. A total of 43 g of salt was obtained from the first recrystallization. This material was dissolved in 120 mL of hot ethanol and left standing at for 48 hr at room temperature. This provided very few needles. The flask was cooled slowly to 4xc2x0 C. to induce more recrystallization. This mixture containing fine needles was filtered over sintered glass and washed with cold acetone (xe2x88x9260xc2x0 C.). More needles formed in the filtrate after addition of the acetone. This was also collected over sintered glass and washed with cold acetone. The process was repeated until no more solid came out from the filtrate. The clear filtrate was put on the rotovap and solvents were removed until cloudiness formed in the flask. This was then cooled to 4xc2x0 C. to coax out more crystals which was collected as described before. This process of crystallization was repeated until crystallization ceased. A total of 33 g of material was obtained from the second crystallization. This material was subjected to 85 mL of hot ethanol and recrystallized again. This time, a substantial amount of needles came out above room temperature. This was collected and the filtrate treated as described before for the previous recrystallizations to provide 29.8 g of a free-flowing material after the third recrystallization. This white powder was dissolved in 78 mL of hot ethanol for the fourth recrystallization. This time, needles came out at room temperature. The fine needles (15 g) were harvested and the filtrate treated as previously described, to provide a total of 27.6 g of a white powder after pumping. The needles were crushed before pumping as they had a tendency to impregnate ethanol. 40 mg of this material was treated with 0.5 mL of a 3.85 M ethyl acetate solution of hydrogen chloride (g) to liberate the acid. The amine salt was removed by trituration with ether followed by filtration over sintered glass. The acid thus obtained had the following optical activity: [xcex1]20D=xe2x88x929.49 (c=0.78, MeOH), [xcex1]20578=xe2x88x9210.38 (c=0.78, MeOH). For use in subsequent reactions, the 2(R)-methyl tetrahydrofuran-2-carboxylic acid was liberated from the salt using a solution of hydrogen chloride(g) in ethyl acetate.
Step A. N-tert-Butoxycarbonyl-(S)4-hydroxyphenylglycine
To a solution of (S)-(4-hydroxyphenyl)glycine (Sigma Chemical) (6.5 g, 39 mmol) in dioxane/water (1:1, 120 mL) was added triethylamine (5,9 g, 8.2 mL, 58 mmol) and [2-(tert-butoxycarbonyloxyimnino)-2-phenylacetonitrile] (BOC-ON; 11 g, 45 mmol). After stirring overnight at room temperature, 300 mL of brine was added to the solution and the mixture was extracted with ether. (3xc3x97100 mL). The aqueous layer was acidified with HCl (pH=2) and extracted with 3xc3x97100 mL of ethyl acetate. The ethyl acetate layer was dried over MgSO4, filtered and the solvent removed under reduced pressure. The residue was chromatographed with 98/2 to 95/5 methylene chloride/methanol. Recovered 12 g of crude product. The impurity was removed following esterification of the product in the next step.
400 MHz 1H NMR (CDCl3): xcex4 1.37 (s, 9H), 5.1 (1H, br s), 6.7 (d, 2H, J=8 Hz), 7.15 (d, 2H, J=8 Hz).
Step B. N-tert-Butoxycarbonyl-(S)-4-hydroxyphenylglycine, Methyl Ester
In a 50 mL round bottomed flask was added a 1:1 mixture of benzene:methanol and N-tert-butoxycarbonyl-(S)-4-hydroxyphenylglycine (2.8 g, 11 mmol). The solution was cooled to 0xc2x0 C. and a 2 M solution of trimethylsilyldiazomethane (Aldrich Chemical Co.) in hexane was added with vigorous stirring until a slight yellow color persisted. Then the reaction mixture solvents were removed under reduced pressure and the crude product was purified by flash chromatography (80/20 hexane/ethyl acetate) to give N-tert-butyloxycarbonyl-(S)-4-hydroxyphenylglycine, methyl ester (2.05 g, 7.3 mmol) (66% yield).
300 MHz 1H NMR (CDCl3): xcex4 1.43 (s, 9H), 3.71 (s, 3H), 5.22 (br d, 1H), 5.57 (1H, br d), 5.80 (br s, 1H), (6.7 (d, 2H, J=8 Hz), 7.17 (d, 2H, J=8 Hz).
Step C. N-tert-Butoxycarbonyl-(S)-4-trifluoromethylsulfonyloxyphenylglycine, Methyl Ester
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butyloxycarbonyl-(S)-4-hydroxyphenylglycine, methyl ester (1.9 g, 6.8 mmol) and pyridine (2.8 mL, 33 mmol) in 12 mL of methylene chloride. The flask was purged with N2, cooled to 0xc2x0 and trifluoromethanesulfonic anhydride (1.38 mL, 7.8 mmol) was added dropwise over several minutes, keeping the temperature at or below 4xc2x0 C. The solution was stirred for 1 h, then at room temperature for 4 h. The mixture was diluted with 20 mL of methylene chloride. The mixture was washed with 20 mL of 0.5 N sodium hydroxide, 1xc3x9720 mL of water and 2xc3x9720 mL of 10% citric acid. The organic layer was dried over anhydrous magnesium sulfate, filtered, and the solvent removed by rotoevaporation. Flash column chromatography on silica gel. eluted with 75/25 hexane/methylene chloride gave 2.3 g of the desired product (81% yield).
300 MHz 1H NMR (CDCl3): xcex4 1.43 (s, 9H), 3.74 (s, 3H), 5.35 (1H, br d), 5.68 (br s, 1H), 7.27 (d, 2H, J=8 Hz), 7.47 (d, 2H, J=8 Hz).
Step D. N-tert-Butoxycarbonyl-(S)-(4-biphenyl)glycine.
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butyloxycarbonyl-(S)-4-trifluoromethylsulfonyloxyphenylglycine, methyl ester (690 mg, 1.67 mmol), anhydrous potassium carbonate (348 mg, 2.6 mmol) and benzeneboronic acid (411 mg, 3.4 mmol) in 15 ml of toluene and 3 mL of ethanol. The mixture was degassed under nitrogen with three freeze-thaw cycles and tetrakis(triphenylphosphine) palladium (94 mg, 0.085 mmol) was added to the reaction mixture and the mixture was heated between 75-90xc2x0 C. for 4 h. The solvent was removed under reduced pressure and the residue purified by flash column chromatography eluted with 85/15 hexane/ethyl acetate. Recovered 600 mg of the methyl ester (quantitative yield).
300 MHz 1H NMR (CDCl3): xcex4 1.44 (s, 9H), 3.75 (s, 3H), 5.37 (1H, br d), 5.62 (br s, 1H), 7.36 (m, 1H), 7.45 (m, 4H), 7.57 (m, 4H).
The ester was hydrolyzed with 1.2 eq of potassium hydroxide in 10 mL of 4:1 ethanol: water (2 h). The solution was acidified with 2 N HCl (pH=2). The solvents were removed in vacuo and the free acid extracted with methylene chloride. 430 mg of the desired free acid was recovered (66% yield).
Step E. 3(S)-(4-Biphenyl)-3-(N-tert-butyloxycarbonyl)amino-1-diazo-propan-2-one.
To a 25 mL round bottom flask fitted with a stir bar and septum was added N-tert-butoxycarbonyl-(S)-4-biphenylglycine (430 mg, 1.31 mmol) in 10 mL of 2:1 methylene chloride: ether. The mixture was cooled to 0xc2x0 C. and N-methyl-morpholine (159 xcexcL, 1.44 mmol) was added, followed by dropwise addition of isobutylchloroformate (179 xcexcL, 1.38 mmol). The mixture was stirred for 1 h at 0xc2x0 C., then diazomethane in ether (excess, prepared from Diazald(copyright) by literature procedures) was added dropwise to the reaction mixture. The mixture was stirred for 1 h then quenched with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate. (2xc3x975 mL), washed with brine then dried over anhydrous magnesium sulfate. The mixture was filtered, the solvent removed under reduced pressure and the product isolated by flash column chromatography on silica gel eluted with 80/20 hexane/ethyl acetate to give 280 mg (0.78 mmol) of desired product (58% yield).
300 MHz 1H NMR (CDCl3): xcex4 1.42 (s, 9H), 5.22 (bs, 1H), 5.29 (s, 1H), 5.9 (br s, 1H), 7.35-7.5 (m, 5H), 7.52-7.62 (m, 4H).
Step F. 3(R)-Amino-3-(4-biphenyl)propionic Acid, Methyl Ester
To a 25 mL round bottom flask fitted with a stir bar and septum was added 3(S)-(4-biphenyl)-3-(N-tert-butyloxycarbonyl)amino-1-diazo-propan-2-one (280 mg, 0.76 mmol), with 5 mL each of methanol and dioxane. The flask was cooled to 0xc2x0 C. and 0.15 eq (34 mg, 0.038 mmol) of silver benzoate in 500 xcexcL of triethylamine was added dropwise to the reaction mixture and the mixture allowed to stir at 25xc2x0 C. for 1 h. The reaction was worked up with 10% ammonium hydroxide in saturated ammonium chloride (10 mL). Extract with ether (3xc3x9710 mL) and dry the organic layer over MgSO4. Filter, reduce the volume and flash chromatograph with 85/15 hexane/ethyl acetate. Recovered 260 mg of product (98% yield). Take this material and dissolve it in 10 mL of 1 N hydrochloric acid in ethyl acetate. After stirring 2 h at room temperature, 180 mg of 3(R)-amino-(4-biphenyl)propionic acid, methyl ester hydrochloride was obtained.
300 MHz 1H NMR (CD3OD): xcex4 2.90 (dd, 1H, J=18 Hz, J=6 Hz), 3.02 (dd, 1H, J=18 Hz, J=6 Hz), 3.66 (s, 3H), 5.9 (br s, 1H), 7.33-7.5 (m, 5H), 7.55-7.6 (m, 4H).
Step A. 4-Benzyloxyphenyldiazoniumtetrafluoroborate.
In a 250 mL round bottomed flask fitted with a stir bar was added 4-benzyloxyaniline (8.7 g, 43.6 mmol), 150 mL of ethanol and 17 mL of 48% fluoroboric acid. Cool to 0xc2x0 C. Then isoamyl nitrite (6,64 mL, 50 mol) was added dropwise over 15 minutes, keeping the solution temperature below 8xc2x0 C. Stir 2 h at 0-4xc2x0 C. The product precipitated out of solution. Diluted the reaction mixture with 100 mL ether and filter the reaction mixture. Wash the precipitate with 2xc3x9750 mL of ether. Recovered 10.3 g (79%) of product. Melting point=137xc2x0 (dec), Lit.=140-142xc2x0 (dec).
Step B. 4-Benzyloxycinnamic Acid, Methyl Ester
The following reaction was adapted from M. Beller and K. Kuhlein, Synlett, p 441 (1995). In a 50 mL round bottomed flask fitted with a stir bar and septum was added 4-benzyloxyphenyldiazoniumtetrafluoroborate (3.0 g, 10.2 mmol) and methyl acrylate (1.72 g, 0 mmol) in 15 mL of methanol. Subsequently, 10% palladium on carbon (250 mg, 0.2 mmol) was added to the mixture and it was heated at 55-60xc2x0 C. until nitrogen gas evolution ceased (2 h) then overnight at 50xc2x0 C. The reaction was cooled to room temperature, the catalyst filtered off and washed with methanol. The solvent is removed under reduced pressure and the residue purified by flash chromatography (90/10 hexane/ethyl acetate) Recovered 2.0 g of the desired product (70% yield).
400 MHz 1H NMR (CDCl3): xcex4 3.78 (s, 3H), 5.08 (s, 2H), 6.25 (d, 1H, J=17 Hz), 6.29 (d, 1H, J=9 Hz), 7.3-7.4 (m, 5H,), 7.45 (d, 2H, J=9 Hz), 7.62 (d, 1H, J=14 Hz).
Step C. 3-(4-benzyloxyphenyl)-3(R)-[benzyl-(1(S)-phenylethyl)-amino]-propionic Acid, Methyl Ester
This procedure was adapted from S. G. Davies and O. Ichihara, Tetrahedron: Asymmetry, 2, p 183 (1991). In a 100 mL round bottom flask fitted with a stir bar and rubber septum is added (S)-(xe2x88x92)-N-benzyl-1-phenylethylamine (1.69 g, 8.0 mmol) in 60 mL of anhydrous tetrahydrofuran. Cooled to 0xc2x0 C. and flushed with nitrogen. n-Butyl lithium (2.5N solution in hexane, 3.2 mL) was added dropwise, keeping the temperature below 4xc2x0 C. for 15 minutes after final base addition. Then cooled to xe2x88x9278xc2x0 C. and slowly added 4-benzyloxycinnamic acid, methyl ester (1.07 g, 4.0 mmol) in 15 ml of dry tetrahydrofuran at such a rate that the solution temperature remaines below xe2x88x9260xc2x0 C. Stirred for 15 minutes, then quenched with saturated ammonium chloride (5 mL). Warmed to room temperature and added 10 mL of saturated brine. Extracted with 2xc3x9725 mL of ether, dried over anhydrous magnesium sulfate. Filtration and evaporation gave a mixture of the adduct and excess amine as a pale yellow oil. Flash column chromatography on silica gel eluted with 90/10 hexane/ethyl acetate gave the desired product (1.25 g, 2.62 mmol) (66% yield.
400 MHz 1H NMR (CDCl3): xcex4 1.19 (d, 2H, J=7 Hz), 2.50 (dd, 1H, J=13 Hz, J=10 Hz), 2.64 (dd, 1H J=13 Hz, J=6 Hz), 3.44 (s, 3H), 3.62 (q, 2H, J=15 Hz), 3.97 (q, 1H, J=6 Hz), 4.36 (dd, 1H, J=9 Hz, J=6 Hz), 5.03 (S, 2H), 6.93 (d, 2H, J=9 Hz), 7.2-7.5 (m, 17 H).
Step D. 3(R)-Amino-3-(4-hydroxphenyl)propionic Acid, Methyl Ester, Acetic Acid Salt
To a 250 mL medium pressure Parr hydrogenation bottle was added 25 mL of methanol, 1 mL of glacial acetic acid, 100 mg of 10% palladium hydroxide on carbon and 3-(4-benzyloxyphenyl)-3(R)-[benzyl-(1(S)-phenylethyl)-amino]propionic acid, methyl ester (1.25 g, 2.6 mmol). The flask was evacuated then pressurized to 50 psi H2. and shaken until no more H2 uptake was observed (4 h). Filter the solution through Celite, wash the pad with methanol (50 mL) and concentrate the filtrate under reduced pressure. Recovered 660 mg of product (theoretical) which was used without further purification.
Magnesium metal (0.6 g, 24.4 mmol) was placed into a 25 mL round bottomed flask and was vigorously stirred under nitrogen for 24 hours. To this activated magnesium was added 1 mL of tetrahydrofuran and 4-bromobenzotrifluoride (0.5 g, 2.2 mmol) and the reaction was sonicated for 30 seconds during which time a deep red color formed. The solution was cooled to 0xc2x0 C. and 3-bromobenzotrifluoride (4.5 g, 20 mmol) in 9 mL of tetrahydrofuran was added dropwise. The reaction was allowed to warm to room temperature and stirred for 1.5 hours then added to a solution of 4-chlorobutyryl chloride (9.4 g, 66.6 mmol) in 50 mL of tetrahydrofuran at xe2x88x9278xc2x0 C. The reaction was allowed to warm to room temperature and stirred for 1 hour. The reaction was washed with water (50 mL), saturated aqueous sodium chloride (50 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography (Biotage) over silica gel eluting with hexane/ethyl acetate (50:1) to give 2.3 g of the title compound as an oil. 1H NMR (500 MHz, CDCl3): xcex4 8.08 (d, 2H, J=8.3 Hz); 7.74 (d, 2H, J=8.0 Hz); 3.69 (m, 2H); 3.21 (m, 2H); 2.25 (m, 2H).
The following compounds were prepared by the procedures described in Reference Example 13 substituting the appropriate aryl bromide, 4-chloro-3xe2x80x2-trifluoromethylbutyrophenone and 4-chloro-3xe2x80x2,4xe2x80x2-bis(trifluoromethyl)-butyrophenone.
Step A. 2-(4-Chlorophenyl)-2-cyanotetrahydrofuran
Potassium cyanide (1.87 g, 28.78 mmol) was added to a solution of 4-chloro-4xe2x80x2-chloro-butyrophenone (5.00 g, 23.03 mmol) in 25 mL of methanol. After stirring for 3 days at room temperature, the reaction was poured into water (50 mL) and extracted with methylene chloride (3xc3x9750 mL). The organic layers were combined, washed with saturated aqueous sodium chloride (50 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography (Biotage) over silica gel eluting with hexane/ethyl acetate (30:1) to give 3.52 g of the title compound as an oil. 1H NMR (500 MHz, CDCl3): xcex4 7.47 (m, 2H); 7.38 (m, 2H); 4.24 (m, 2H); 2.74 (m, 1H); 2.33 (m, 1H), 2.15 (m, 2H).
Step B. 2-(4-Chlorophenyl)-2-tetrahydrofuroic Acid.
Potassium hydroxide (4.7 g, 84.9 mmol) was added to a solution of 2-(4-chlorophenyl)-2-cyanotetrahydrofuran (3.53 g, 16.98 mmol) in 25 mL of ethylene glycol. The reaction was stirred at 195xc2x0 C. for 2.5 hours. The reaction was cooled and extracted with 25 mL of methylene chloride, which was discarded. The aqueous layer was made acidic with concentrated HCl. and extracted with methylene chloride (3xc3x9725 mL). These organics were combined, washed with saturated aqueous sodium chloride (25 mL), dried over MgSO4 and concentrated in vacuo. Recrystallization from hexanes yielded 2.87 g of the title compound as a white crystalline solid. 1H NMR (500 MHz, CDCl3): xcex4 11.15 (bs, 1H), 7.50 (d, 2H, J=8.4 Hz); 7.30 (d, 2H, J=8.5 Hz); 4.13 (q, 1H, J=7.5 Hz); 4.05 (q, 1H, J=7.5 Hz); 2.79 (m, 1H); 2.20 (m, 1H), 1.99 (m, 1H); 1.95 (m, 1H). 13C NMR (125 MHz, CDCl3): xcex4 177.1, 138.6, 133.8, 128.3, 126.9, 86.7, 69.3, 37.4, 25.3.
The following compounds were prepared by the procedures described in Reference Example 14 substituting the appropriate butyrophenone: 2-(4-bromophenyl)-2-tetrahydrofuroic acid, 2-(4-tert-butylphenyl)-2-tetrahydrofuroic acid, 2-(4-trifluoromethylphenyl)-2-tetrahydrofuroic acid, 2-(3-trifluoromethylphenyl)-2-tetrahydrofuroic acid, 2-(3,5-bis(trifluoromethyl)phenyl)-2-tetrahydrofuroic acid, 2-(3,4-dimethylphenyl)-2-tetrahydrofuroic acid, 2-cyclohexane-2-tetrahydrofuroic acid, 2-(3-nitrophenyl)-2-tetrahydrofuroic acid, and 2-(3-nitro-4-chlorophenyl)-2-tetrahydrofuroic acid.
Step A. 2-Carbobenzyloxytetrahydrothiophene 1,1-Dioxide
Tetramethylene sulfone (2.00 g, 16.64 mmol) and benzyl chloroformate (3.12 g, 18.30 mmol) were dissolved in 50 mL of tetrahydrofuran and cooled to xe2x88x9278xc2x0 C. Lithium bis(trimethylsilyl)amide (33.2 mL, 33.2 mmol, 1.0M in tetrahydrofuran) was added to the mixture dropwise. The reaction was stirred for 30 minutes at xe2x88x9278xc2x0 C. then allowed to warm to room temperature and stirred for 30 minutes. The reaction was quenched with 1N HCl (50 mL) and extracted with methylene chloride (3xc3x9730 mL). The combined extracts were washed with saturated aqueous sodium chloride (50 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography (Biotage) over silica gel eluting with hexane/ethyl acetate (3:1) to give 2.70 g of the title compound as a colorless oil. 1H NMR (500 MHz, CDCl3): xcex4 7.20 (m, 5H); 5.29 (d, 1H, J=12.1 Hz); 5.23 (d, 1H, J=12.2 Hz); 3.95 (t, 1H, J=7.8 Hz); 3.12 (m, 2H); 2.85 (m, 1H); 2.36 (m, 2H); 2.16 (m, 1H). 13C NMR (125 MHz, CDCl3): xcex4 165.4, 134.8, 128.58, 128.55, 128.45, 68.3, 64.7, 51.5, 26.0, 20.4.
Step B. 2-Carbobenzyloxy-2-methyltetrahydrothiophene 1,1-Dioxide
2-Carbobenzyloxytetrahydrothiophene 1,1-dioxide (2.70 g, 10.62 mmol) and iodomethane (0.7 mL, 11.2 mmol) were dissolved in 200 mL of tetrahydrofuran and cooled to xe2x88x9278xc2x0 C. Lithium bis(trimethylsilyl)amide (11.2 mL, 1.2 mmol, 1.0M in tetrahydrofuran) was added to the mixture dropwise. The reaction was stirred for 1 hour at xe2x88x9278xc2x0 C. then allowed to warm to room temperature and stirred for 1 hour. The reaction was quenched with 1N HCl (25 mL) and extracted with methylene chloride (3xc3x9725 mL). The combined extracts were washed with saturated aqueous sodium chloride (25 mL), dried over MgSO4 and concentrated in vacuo. The residue was purified by flash chromatography (Biotage) over silica gel eluting with hexane/ethyl acetate (2:1) to give 2.14 g of the title compound as a pale yellow oil. 1H NMR (500 MHz, CDCl3): xcex4 7.36 (m, 5H); 5.27 (d, 1H, J=12.4 Hz); 5.20 (d, 1H, J=12.4 Hz); 3.21 (td, 1H, J=13.2, 8.1 Hz); 3.11 (ddd, 1H, J=5.9, 8.0, 13.7 Hz)); 2.83 (dt, 1H, J=7.1, 13.8 Hz); 2.28 (m, 1H); 2.10 (m, 1H); 1.98 (m, 1H): 1.62 (s, 3H). 13C NMR (125 MHz, CDCl3): xcex4 168.4, 135.0, 128.6, 128.4, 128.2, 68.2, 67.1, 50.7, 34.0, 18.8, 18.3.
Step C. 2-Methyltetrahydrothiophene 1,1-Dioxide-2-carboxylic Acid
2-Carbobenzyloxy-2-methyltetrahydrothiophene 1,1-dioxide (2.14 g, 7.98 mmol) and palladium on carbon (10%, 1 g) were dissolved in 20 mL of ethyl acetate. The reaction was placed under a balloon of hydrogen gas and stirred for 16 hours. The reaction was filtered and concentrated in vacuo to give 1.07 g of the title compound as a white solid. 1H NMR (500 MHz, CD3OD): xcex4 3.2 (m, 2H); 2.77 (dt, 1H, J=7.3, 14.2 Hz); 2.22 (m, 1H); 2.10 (m, 1H); 1.98 (m, 1H): 1.56 (s, 3H). 13C NMR (125 MHz, CD3OD): xcex4 171.2, 68.3, 51.6, 34.8, 19.5, 19.0.
The following compounds were prepared by the procedure described in Example 15 substituting the appropriate sulfone: 2-methylthietane 1,1-dioxide-2-carboxylic acid, 2-methyl-1,4-thioxane-1,1-dioxide-2-carboxylic acid.
Step A. 2-Methyl-4-methylene-2-tetrahydrofuroic Acid, Methyl Ester
At 0xc2x0 C., to a solution of R(+)-methyl lactate (4.16 g, 40 mmole) and 1,1-bis(chloromethyl)ethene (5.00 g, 40 mmole) in 100 mL of DMF was added NaH (2.64 g, 110 mmole) in several batches at such a rate as to avoid overreaction. After 10 min, the ice bath was removed and the reaction mixture was allowed to stir at room temperature overnight. It was diluted with ether and poured into a mixture of 80 mL saturated aqueous ammonium chloride and ice. The phases were separated. Further extractions of the aqueous phase with ether twice was followed by washing the combined organic layers with water, brine, and drying over anhydrous magnesium sulfate. Careful removal of volatiles provided 6 g of crude product as a mixture of the desired compound and the endo-double bond isomer. Due to its volatility, chromatographic separation was not attempted. Atmospheric pressure distillation (bp 60xc2x0 C.) provided 0.8 g of desired product. The product readily decomposed under heat. TLC: Rf=0.5 in 4:1 hexane:ethyl acetate.
500 MHz 1H NMR (CDCl3): xcex4 1.53(s, 3H), 2.52(dm, J=15 Hz, 1H),.2.98 (dm, J=15 Hz, 1H), 3.75(s, 3H), 4.47(br s, 2H), 4.93(m, 1H), 5.01(m, 1H).
Step B. 2-Methyl-4-methyl-2-tetrahydrofuroic Acid
To a solution of 2-methyl-4-methylene-2-tetrahydrofuroic acid, methyl ester (156 mg, 0.5 mmol, obtained from Step A) in 0.25 mL of methanol was added 6 mg of 10% platinum oxide and hydrogenated at room temperature for 2 h. TLC indicated disappearance of all starting material and formation of a single new spot. Since this compound is expected to be very volatile, it was not isolated but used as the crude mixture from hydrogenation in the next reaction where the product would be an acid and be much less volatile.
The above reaction mixture was treated with 1.5 mmole of sodium hydroxide (0.12 mL of 12.5N NaOH) and allowed to stir at room temperature for 6 h. TLC indicated that all starting material was gone. After removal of the volatiles, the residue was treated with excess hydrogen chloride in ethyl acetate. The excess acid was removed by a stream of nitrogen, and the residue was pumped, pumped with methanol 3 times (to get rid of water). This provided 50 mg of a white solid. The NMR suggested this to be two pairs of diastereomers in 1:2 ratio. TLC: Rf=0.2 in 10:1 dichloromethane:methanol
500 MHz 1H NMR (CDCl3): xcex4 0.92, 1.03 (d, J=6.7 Hz, 3H), 1.42, 1.47(s, 3H), 1.75(dd, J=12.6, 1.8 Hz, 1H), 2.11 (dd, J=12.4, 7.1 Hz, 1H), 2.55 (m, 1H), 3.55, 3.75(t, J=8.3 Hz, 1H), 4.05, 4.16(t, J=8.0 Hz, 1H).
Step A. 2-Methyl-4-methylene-2-tetrahydrofuroic Acid, tert-Butyl Ester
At 0xc2x0 C., to a solution of R(+)-tert-butyl lactate (2.0 g, 13.7 mmole) and 1,1-bis(chloromethyl)ethene (1.71 g, 13.7 mmole) in 33 mL of DMF was added NaH (0.69 g, 29 mmole) all at once. After 10 min, the ice bath was removed and the reaction mixture was allowed to stir at room temperature overnight. It was poured into a mixture of ice and ether. The phases were separated. Further extractions of the aqueous phase with ether twice was followed by washing the combined organic layers with water, brine, and drying over anhydrous magnesium sulfate. Volatiles were removed at low temperature under reduced pressure and the crude product was flash chromatographed over silica gel (gradient elution using 60-10/1 petroleum ether/ether) to provide 1.05 g of the desired compound as a clear liquid (39%), homogeneous by TLC (Rf=0.65 in 4:1 hexane:ethyl acetate); Mass Spectrum: EI m/e 198 (M+), 142 (M-tBu)+.
500 MHz 1H NMR (CDCl3): xcex4 1.45 (s, 9H), 1.47(s, 3H), 2.44(d, J=5.8 Hz, 1H), 2.90 (d, J=5.7 Hz, 1H), 4.45 (m, 2H), 4.89(m, 1H), 4.97 (m, 1H).
At 0xc2x0 C., to a solution of 2-methyl-4-methylene-2-tetrahydrofuroic acid, tert-butyl ester dissolved in 0.3 mL dichloromethane was added 461 mL of a 1:1 solution of trifluoroacetic acid and dichloromethane. After 20 min, the ice bath was removed and the reaction mixture allowed to stir at room temperature for 5 h. The excess trifluoroacetic acid was removed by a stream of nitrogen, and the residue coevaporated with dichloromethane 3 times. The crude product was chromatographed via silica gel (gradient elution 0-12% MeOH/CH2Cl2) to give 55 mg of the desired acid as a glass (77%). Mass Spectrum: EI m/e 142 (M)+.
500 MHz 1H NMR (CDCl3): xcex4 1.57 (s, 3H), 2.58(dm, J=16 Hz, 1H), 2.62 (dm, J=16 Hz, 1H), 4.51 (br s, 2H), 4.99(m, 1H), 5.06 (m, 1H).
Step A. 2-Methyl-4-hydroxymethyl-2-tetrahydrofuroic Acid, tert-butyl Ester
2-Methyl-4-methylene-2-tetrahydrofuroic acid, tert-butyl ester (200 mg, 1 mmole) was dissolved in 4 mL of tetrahydrofuran, cooled to 0xc2x0 C., and boranetetrahydrofuran complex (2.5 mL of a 1.0 M solution in tetrahydrofuran) was added dropwise via a hypodermic syringe. The ice bath was removed and the reaction mixture was allowed to stir at room temperature for 1.5 h when TLC (1/1 hexane/ethyl acetate) indicated almost complete disappearance of the starting material. The mixture was cooled to 0xc2x0 C. again and treated dropwise with 1 mL of 30% aqueous hydrogen peroxide. After being stirred for 2 h at room temperature, the reaction mixture was diluted with ether and washed with 5% sodium bicarbonate. The phases were separated and the aqueous phase was reextracted with ether twice. The organic layers were combined and washed with water and brine and dried over anhydrous sodium sulfate. The crude product obtained after filtration and removal of volatiles was chromatographed (gradient elution using 4-1/1 hexane/ethyl acetate) to provide 120 mg (56%) of two pairs of diastereomers, inseparable cleanly by column chromatography. TLC: Rf=0.15 (4:1 hexane:ethyl acetate)
500 MHz 1H NMR (CDCl3): xcex4 1.45 (2s, 3H), 1.49, 1.50 (2s, 9H), 1.54, 1.94 (2dd, J=13.1, 9.2 Hz, 1H), 2.14, 2.47(2dd, J=13, 6.2 Hz; 13, 8.2 Hz, 1H), 2.56 (m, 1H), 3.62 (m, 2H), 3.76, 3.80 (2dd, J=9.0, 7.4 Hz, 8.7, 5.5 Hz, 1H), 4.10(m, 1H).
Step B. 2-Methyl-4-(pyrrolidine-1-carbonyloxymethyl)-2-tetrahydrofuroic Acid, tert-Butyl Ester
2-Methyl-4-hydroxymethyl-2-tetrahydrofuroic acid, tert-butyl ester (60 mg, 0.277 mmol, from Step A) was solved in 0.5 mL of pyridine and p-nitrochloroformate (61.5 mg, 0.305 mmole) was added. After being stirred at room temperature for 5 h, pyrrolidine (0.042 mL, 0.5 mmole) was added all at once. The yellow reaction mixture was stirred at room temperature overnight. The reaction mixture was then diluted with ethyl acetate, washed with 5% citric acid, water, brine, and dried over anhydrous magnesium sulfate. The crude product obtained after filtration and removal of volatiles was flash chromatographed via silica gel (gradient elution using 40-10/1 hexane/ethyl acetate). This provided a fraction (43 mg) containing mainly the desired product. This was used without further purification in the next reaction. TLC: Rf=0.5 (4:1 hexane:ethyl acetate).
500 MHz 1H NMR (CDCl3): xcex4 1.44 (s, 3H), 1.48, (2s, 9H), 1.96 (br m, 4H), 1.56, 2.0 (dd, J=13, 8.9 Hz, 12.8, 4.5 Hz, 1H), 2.10, 2.46 (2dd, J=13.1, 8.3 Hz; 13, 7.3 Hz, 1H), 2.8 (m, 1H), 3.35, 3.40 (2m, 4H), 3.72, 3.79 (2m, 1H), 4.0 (m, 1H), 4.10(m, 2H).
Step C. 2-Methyl-4-(pyrrolidine-1-carbonyloxymethyl)-2-tetrahydrofuroic Acid
2-Methyl-4-(pyrrolidine-1-carbonyloxymethyl)-2-tetrahydrofuroic acid, tert-butyl ester (from Step B) was hydrolyzed as described in Reference Example 17, Step B to provide the title compound in a 72% yield after chromatographic purification. TLC: Rf=0.2 (10:1 dichloromethane:methanol)
500 MHz 1H NMR (CDCl3): xcex4 1.44, 1.48 (2s, 3H), 1.88 (br m, 4H), 1.63, 2.06 (dd, J=13.1, 8.3 Hz, 13.1, 9.0 Hz, 1H), 2.17, 2.52 (2dd, J=13, 8 Hz; 13, 7 Hz, 1H), 2.68 (m, 1H), 3.34 (m, 4H), 3.72(m, 1H), 3.98-4.10 (m, 3H).
Step A. 2-methyl-4-azidomethyl-2-tetrahydrofuroic Acid, tert-butyl Ester
2-Methyl-4-hydroxymethyl-2-tetrahydrofuroic acid, tert-Butyl Ester (400 mg, 1.85 mmol, from Reference Example 18, Step A) was dissolved in 7.5 mL of toluene, and triethylamine (1.85 mmol, 187 mg) was added dropwise. After cooling to 0xc2x0 C., methanesulfonyl chloride (1.85 mmol, 212 mg) was added and stirring was continued for 20 min. Subsequently, tetra-n-butylammonium bromide (1.85 mmol, 596 mg) and excess aqueous sodium azide (15.7 mmol, 1.02 g, dissolved in 3.7 mL water) was added. The mixture was stirred at 60xc2x0 C. for 8 h. TLC (4/1 hexane/ethyl acetate) indicated the formation of new spot and the disappearance of most of the starting material. After being cooled to room temperature, the reaction mixture was diluted with ether and citric acid was added to attain pH5. The phases were separated and the aqueous phase was reextracted with ether twice. The organic layers were combined and washed with brine and dried (MgSO4). The residue obtained after filtration and removal of most of the volatiles was flash chromatographed (silica gel, gradient elution 50-20/1 hexane/ethyl acetate) to provide 280 mg (63%) of the azide. TLC: Rf=0.8 (4:1 hexane:ethyl acetate) 500 MHz 1H NMR (CDCl3): xcex4 1.45 (s, 3H), 1.49, 1.50 (2s, 9H), 1.55, 2.0 (2dd, J=12.8, 8.0 Hz, 13.1, 9.0 fHz, 1H), 2.12, 2.50 (dd, J=13.1, 6.4 Hz, 12.8, 8.0 Hz, 1H), 2.60 (m, 1H), 3.35 (m, 2H), 3.68, 3.73(2dd, J=8.7, 5.7 Hz, 8.7, 7.5 Hz, 1H), 4.10 (m, 1H).
Step B. 2-Methyl-4-aminomethyl-2-tetrahydrofuroic Acid, tert-Butyl Ester
At room temperature, 2-methyl-4-azidomethyl-2-tetrahydrofuroic Acid, tert-butyl ester (330 mg, 1.37 mmol, from Step A) was dissolved in tetrahydrofuran (2.75 mL) and triphenylphosphine (1.40 mmol, 367 mg) was added batchwise over 10 min. After being stirred at room temperature for 2 h, water (2.1mmol, 0.038 mL) was added dropwise via a hyperdermic syringe. Stirring was continued at room temperature for 24 h when TLC indicated disappearance of all starting material. The volatiles were removed and the crude product was flash chromatographed to provide 198 mg (67%) of the desired amine as a clear oil. TLC: Rf=0.1 (4:1 hexane:ethyl acetate).
500 MHz 1H NMR (CDCl3): xcex4 1.40 (2s, 3H), 1.45(2s, 9H), 2.0 (m, 1H), 2.35 (m, 1H), 2.48 (m, 1H), 2.70 (m, 2H), 3.65(m, 1H), 4.10 (m, 1H).
Step C. 2-Methyl-4-benzoylaminomethyl-2-tetrahydrofuroic Acid, tert-Butyl Ester
At room temperature, to a solution of 2-methyl-4-aminomethyl-2-tetrahydrofuroic acid, tert-butyl ester (30 mg, 0.139 mmole) was added benzoyl chloride (0.167 mmol, 24 mg) and N-methylmorpholine (0.334 mmol, 34 mg). After being stirred at room temperature for 2.5d, volatiles were removed and the residue was flash chromatographed through silica gel. The title compound was obtained in quantitative yield (47 mg). TLC: Rf=0.45 (1:1 hexane:ethyl acetate); Mass Spectrum: ESI m/e 320 (M+1)+.
500 MHz 1H NMR (CDCl3): xcex4 1.44, 1.49 (2s, 3H), 1.46, 1.47 (2s, 9H), 1.58, 2.02 (2m, 1H), 2.16, 2.53 (dd, J=13.3, 6.4 Hz, 12.8, 8.0 Hz, 1H), 2.70 (m, 1H), 3.48 (m, 2H), 3.76(m, 1H), 4.06 (m, 1H), 6.5, 6.7 (2 br s, 1H), 7.45(m, 2H), 7.50(m, 1H), 7.78(m, 2H).
Step D. 2-methyl-4-benzoylaminomethyl-2-tetrahydrofuroic Acid
2-Methyl-4-benzoylaminomethyl-2-tetrahydrofuroic acid, tert-butyl ester was hydrolyzed according to the procedure of Reference Example 17, Step B to provide the title compound. TLC: Rf=0.3 (10:1 dichloromethane:methanol); Mass Spectrum: ESI m/e 264 (M+1)+.
500 MHz 1H NMR (CDCl3): xcex4 1.50, 1.56 (2s, 3H), 1.70, 2.15 (2m, 1H), 2.24, 2.62 (2m, 1H), 2.72 (m, 1H), 3.50 (m, 2H), 3.80(m, 1H), 4.12 (m, 1H), 6.8, 7.0 (2 br s, 1H), 7.42(m, 2H), 7.54(m, 1H), 7.76(m, 2H), 7,80 (br s, 1H).
The title compound was prepared according to the procedures described in Reference Example 19, substituting benzene sulfonyl chloride for benzoyl chloride in Step C. Mass Spectrum: ESI m/e 300 (M+1)+, 255 (Mxe2x80x94CO2H)+.
Step A. 2-Methyl-4-oxo-2-tetrahydrofuroic Acid, tert-Butyl Ester
A solution of 2-methyl-4-methylene-2-tetrahydrofuroic acid, tert-butyl ester (200 mg, 1 mmole) in 20 mL of 1:1 dichloromethane:methanol was treated with ozone until the ozololysis of the exocyclic methylene was complete. The ozonide was decomposed via dimethylsulfide. Removal of the volatiles provided 198 mg of an oil, homogeneous on TLC (4/1 hexane/ethyl acetate).
500 MHz 1H NMR (CDCl3): xcex4 1.47, (s, 9H), 1.61 (s, 3H), 2.40(d, J=8.1 Hz, 1H), 2.80 (d, J=8.0 Hz, 1H), 4.12 (d, J=15 Hz, 2H).
Step B. 2-Methyl-4-oxo-2-tetrahydrofuroic Acid
2-Methyl-4-oxo-2-tetrahydrofuroic acid, tert-butyl ester (from Step A) was hydrolyzed according to the procedure of Reference Example 17, Step B to provide the title compound in 77% yield after chromatographic purification.
500 MHz 1H NMR (CDCl3): xcex4 1.71 (s, 3H), 2.50(d, J=8.0 Hz, 1H), 2.93 (d, J=8.2 Hz, 1H), 4.18 (m, 2H), 7.25(br s, 1H).
Step A. Methyl 2-(2,6-Dichlorophenyl)-1,3-benzoxazole-5-carboxylate
A solution of 2,6-dichlorobenzaldehyde (1.2 g, 6.8 mmol) and methyl 3-amino-4-hydroxybenzoate (1.0 g, 5.9 mmol) was refluxed in toluene (50 mL) overnight. The mixture was concentrated to dryness (rotary evaporator), and the residue was suspended in acetic acid (25 mL). The mixture was cooled by an ice-water bath, and was added-lead tetraacetate (3.2 g, 7.7 mmol). After stirring at room temperature for 4 h, the mixture was poured into ice water, and the product was extracted with ether (4xc3x9730 mL). The combined extracts were dried over MgSO4, filtered and was concentrated. The residue was purified on a silica gel column eluting with 4:1 hexane/ethyl acetate to give the product (1.2 g, 60%).
Step B. 2-(2,6-Dichlorophenyl)-5-hydroxymethyl-1,3-benzoxazole
To a solution of the carboxylate of Step A (1.2 g, 3.9 mmol) in 30 mL of dry THF was added LiAIH4 (0.34 g, 8.5 mmol) in one portion at xe2x88x9230 C After stirring for 3 h (bath temperature slowly rose to room temperature), the reaction mixture was carefully poured into brine and 2 M HCl. The product was extracted with ethyl acetate (3xc3x9730 mL). The combined extracts were dried over MgSO4, filtered and concentrated to dryness to give the product, which was used immediately for the next step.
Step C. 5-Bromomethyl-2-(2,6-dichlorophenyl)-1,3-benzoxazole
To a suspension of N-bromosuccinimide (1.2 g, 6.7 mmol) in 10 mL of methylene chloride at xe2x88x9220 C was added dimethyl sulfide (0.50 mL, 6.7 mmol). After stirring at 0 C for 5 min, the reaction was cooled to xe2x88x9220 C and was added the alcohol obtained at Step B in 10 mL of methylene chloride. The bath was then removed, and the reaction was stirred at room temperature for 2 h, The reaction mixture was poured into water, and the product was extracted with ether (2xc3x9750 mL). The combined extracts were dried over MgSO4, filtered and concentrated to dryness to give the crude bromide (1.3 g), which was used directly for the next step.
Step D. 3-(2-(2,6-Dichlorophenyl)-1,3-benzoxazol-5-yl)-N-diphenylmethylene)alanine, tert-Butyl Ester
To a solution of the bromide of Step C (1.3 g, 3.7 mmol), t-butyl N-diphenylmethyleneglycinate (1.3 g, 4.4 mmol) and O(9)-allyl-N-9-anthracenylmethylcinchonidium bromide (0.24 g, 0.40 mmol) in 10 mL of methylene chloride at xe2x88x9278 C was added cesium hydroxide mono-hydrate (7.0 g, 41 mmol) (Corey, EJ; et al J. Am. 
Chem. Soc. 1997, 119, 12414). The reaction was stirred for overnight at xe2x88x9250 C. The resulting mixture was poured into water, and the product was extracted with ether 3xc3x9750 mL). The combined extracts were dried over MgSO4, filtered and concentrated. The residue was purified on a silica gel column eluting with 6:1 hexane/ethyl acetate to give the product (2.1 g, 95% from the ester over 3 steps).
1H NMR (500 MHz, CD3OD) xcex4 7.64-7.26 (14H, m), 7.22 (1H, dd, J=8.5, 1.5 Hz), 6.59 (1H, d, J=7.0 Hz), 4.22 (1H, dd, J=9.0, 4.5 Hz), 3.40 (1H, dd, J=13.5, 4.5 Hz), 3.26 (1H, dd, J=9.5, 4.5 Hz), 1.46 (9H, s).
Step E. 3-(2-(2,6-Dichlorophenyl)-1,3-benzoxazol-5-yl)alanine, tert-Butyl Ester, Hydrochloride Salt
To a solution of the ester of Step D (2.1 g, 3.7 mmol) in 5 mL of THF was added acetic acid (5 mL) and water (5 mL) at room temperature. After stirring at room temperature for 2 h, the reaction mixture was poured into saturated sodium bicarbonate and saturated sodium carbonate (50 mL each), and the product was extracted with ethyl acetate (3xc3x9730 mL). The combined extracts were dried over Na2SO4, filtered and concentrated. The residue was purified on a silica gel column eluting with 4:1 hexane/ethyl acetate to 9:1:0.1:0.1 ethyl acetate/hexane/methanol/triethyl amine to give the product, which was converted to the corresponding hydrochloride salt by treatment with 1 M hydrogen chloride in ether (1.2 g, 75%).
1H NMR (500 MHz, CD3OD) xcex4 7.79 (1H, br s), 7.75 (1H, d, J=8.5 Hz), 7.64-7.60 (3H, m), 7.47 (1H, d, J=8.5 Hz), 4.28 (1H, t, J=8.0 Hz), 3.38 (1H, d, J=8.0 Hz), 1.41 (9H, s); LC-MS: calculated for C20H20Cl2N2O3, 406; found m/e 407 (M+H+).
Step A. t-Butyl xcex1-diazoacetoacetate
To a solution of t-butylacetate (11.9 g, 75 mmol) in 100 mL of anhydrous acetonitrile was added triethylamine (7.58 g). At 20xc2x0 C., p-tolueneazide (prepared according to Org. Syn. Col. Vol. V. p 179) was added dropwise over 15 minutes. After being stirred at room temperature for 2.5 h, the volatiles were removed at 35xc2x0 C. under reduced pressure and the residue was triturated with ether, washed with aqueous KOH (4.5 g KOH/50 mL water, then 0.75 g KOH/25 mL water), and water. The organic layer was dried over anhydrous sodium sulfate and the volatiles were removed until constant weight was obtained. This gave 13.7 g of a yellow liquid (quantitative) which was used in the next step without further purification, homogeneous by TLC (Rf=0.8 in 1/1 hexane/ethyl acetate). This procedure is based on Org. Syn. Col.Vol.V. p.179
NMR: 400 MHz 1H NMR (CDCl3) xcex4 1.54 (s, 9H), 2.45 (s, 3H).
Step B. t-Butyl 2-diazo-3-oxo-5-hydroxyhexanoate
At xe2x88x9278xc2x0 C., to a solution of t-butyl xcex1-diazoacetoacetate (0.400 g, 2.17 mmol, obtained from Step A) in 50 mL of dichloromethane was added triethylamine dropwise, followed by dichlorophenylborane (2.64 mmol). The yellow reaction mixture was stirred at xe2x88x9278xc2x0 C. for 3 h. Acetaldehyde (0.3 mL) was added dropwise and stirring was continued for 2 h at xe2x88x9278xc2x0 C. After quenching the reaction at xe2x88x9278xc2x0 C. with 30 mL of a 1/1 methanol/pH 7 buffer, the temperature was raised to 0xc2x0 C. followed by addition of 7 mL of a 1/1 methanol/H2O2 (13% aq) solution. Half an hour later, phases were separated, the aqueous layer was reextracted with dichloromethane twice and the combined organic layers was washed with saturated aqueous sodium bicarbonate and 1M aq. NaOH. The aqueous layer was back extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate. The crude product obtained after filtration and removal of volatiles was purified via flash chromatography over silica gel, eluting with 40-5/1 mixtures of hexane/ethyl acetate. This provided 350 mg of the desired compound as a clear oil (71%), homogeneous by TLC (Rf=0.35, 4/1 hexane/ethyl acetate);
NMR: 400 MHz 1H NMR (CDCl3) xcex4 1.25 (d, J=7.5 Hz, 3H), 1.54 (s, 9H), 2.88(dd, J=17.4, 9.2 Hz, 1H), 3.10 (dd, J=17.5, 2.1 Hz, 1H), 4.28 (m, 1H).
Step C. 5-Methyl-3-keto-2-tetrahydrofuroic Acid, t-Butyl Ester
Rhodium(II) acetate dimer (66 mg) was taken up in 7.5 mL of benzene and heated to 90xc2x0 C. for 15 min. To this hot solution was added dropwise a solution of t-butyl 2-diazo-3-oxo-5-hydroxyhexanoate (from Step B) in 15 mL of dichloroethane. The reaction was completed ten minutes after completion of addition. After being cooled to room temperature, the reaction mixture was filtered over a pad of celite and the crude material obtained after removal of volatiles was passed through a miniBiotage 40 column and eluted using 10/1 hexane/ethyl acetate. This provided 503 mg (82.3%) of the desired material as a white glassy solid, homogenous by TLC (Rf=0.40, 4/1 hexane/ethyl acetate) but is a 3:1 mixture of the 2 pairs of diastereomers as shown by NMR;
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.47, 1.53 (2d, J=6.0 Hz, 3H), 1.49, 1.50 (2s, 9H), 2.21, 2.28 (2dd, J=18, 10 Hz, 1H) 2.64 (dd, J=18, 6 Hz, 1H), 4.32, 4.48 (2s, 1H), 4.41, 4.75(m, 1H).
Step D. 2,5-Dimethyl-3-oxo-2-tetrahydrofuroic Acid, t-Butyl Ester
5-Methyl-3-keto-2-tetrahydrofuroic acid, t-butyl ester (790 mg, 3.95 mmol, from Step C) was dissovled in 1.2 mL of acetonitrile and placed in a sealed tube with a stirring bar. Anhydrous potassium carbonate (552 mg, 4 mmole) was added followed by iodomethane. The mixture was stirred at 90xc2x0 C. for 24 h. After being cooled to room temperature and filtered through sintered glass, the residue was washed with copious amounts of acetonitrile and the volatiles were removed under reduced pressure. The crude product was chromatographed using a Biotage short column and gradient elution (100-20/1 hexane/ethyl acetate). This provided 694 mg (83%) of the desired product as an oil, homogenous by TLC (Rf=0.5 in 4/1 hexane/ethyl acetate). NMR shows this to be a 3:1 distereomeric pair mixture.
NMR: 400 MHz 1H NMR (CDCl3) xcex4 1.47, 1.44 (2d, J=6.0 Hz, 3H), 1.41, 1.44(2s, 3H), 1.47(s, 9H), 2.16, 2.39 (2dd, J=18, 10 Hz, 1H), 2.63, 2.69 (2dd, J=18, 6 Hz, 1H), 4.41, 4.60 (m, 1H).
Step E. 2,5-Dimethyl-3-oxo-2-tetrahydrofuroic Acid
2-Methyl-3-oxo-2-tetrahydrofuroic acid, t-butyl ester (100 mg, 0.5 mmol) was treated with 1/1 trifluoroacetic acid/dichloromethane at 0xc2x0 C. The reaction mixture was stirred at room temperature until all starting material had disappeared by TLC. The excess trifluoroacetic acid was removed via a stream of nitrogen and the residue was coevaporated with dichloromethane and purified via a silica gel SepPak plug. This 30 provided 49 mg of the desired product (68%), homogeneous by TLC (Rf=0.1 in 4/1 hexane/ethyl acetate).
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.47, 1.51 (2d, J=6.2 Hz, 3H), 1.50, 1.54 (2s, 3H), 2.24, 2.41 (2dd, J=18, 9.8 Hz, 1H), 2.73 (dd, J=18, 6 Hz, 1H), 4.46, 4.60 (m, 1H) The following compounds were prepared by the procedure described in Reference Example 23 using the appropriate aldehyde: 2-benzyl-5-methyl-3-oxo-2-tetrahydrofuroic acid, 5-Methyl-3-oxo-2-phenyl-2-tetrahydrofuroic acid.
Step A. 2,5-Dimethyl-3-hydroxy-2-tetrahydrofuroic Acid, t-Butyl Ester
2,5-Dimethyl-3-oxo-2-tetrahydrofuroic acid, t-butyl ester (50 mg, 0.35 mmol, from Reference Example 23, Step D) was dissolved in anhydrous methanol (4 mL) and cooled to 0xc2x0 C. Sodium borohydride (0.39 mmol, 15 mg) was added all at once and the reaction mixture was stirred for another 20 minutes at 0xc2x0 C. After acetone (0.2 mL) was added to quench the reaction mixture, saturated aqueous ammonium chloride (8 mL) was added and the reaction mixture was stirred at for 10 minutes 0xc2x0 C. followed by at room temperature for 30 minutes. Methylene chloride was added to extract the organic material. The aqueous phase was re-extracted twice with dichloromethane. The organic layers were combined and washed with brine and dried over anhydrous sodium sulfate. This provided 53 mg of the title compound (70%), homogeneous by TLC (Rf=0.15 in 4/1 hexane/ethyl acetate).
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.28, 1.31, 1.34 (3d, J=7 Hz, 3H), 1.40, 1.41, 1.49 (s, 3H), 1.46, 1.50 (s, 9H), 1.55-1.80 (m, 1H), 2.30-2.43 (m, 1H), 3.0 (m, 1H), 4.21, 4.42 (2m, 1H), 4.39, 4.43 (2m, 1H).
Step B. 2,5-Dimethyl-3-hydroxy-2-tetrahydrofuroic Acid
The title compound was prepared according to the procedure of Reference Example 23, Step E. After chromatographic separation, 2 fractions were obtained. One of these (Diastereomer 1) is a single pair of compounds, the other one (Diastereomer 2) contains 2 pairs of diastereomers; each homogeneous by TLC (1/1 hexane/ethyl acetate).
Diastereomer Pair 1: NMR: 500 MHz 1H NMR (CD3OD) xcex4 1.27 (m, 1H), 1.34 (3d, J=7.2 Hz, 3H), 1.38 (s, 3H), 1.71 (m, 1H), 2.39 (m, 1H), 4.17 (m, 1H), 4.26(m, 1H). Diastereomer Pairs 2: NMR: 500 MHz 1H NMR (CD3OD) xcex4 1.28, 1.34 (3d, J=6.2 Hz, 3H), 1.38 (s, 3H), 1.52, 1.70 (m, 1H), 2.35, 2.42 (m, 1H), 4.10-4.37 (m, 1H), 4.47(m, 1H),
Step A. 2,5-Dimethyl-3-methoxycarbonylmethylene-2-tetrahydrofuroic Acid, t-Butyl Ester
2,5-Dimethyl-3-oxo-2-tetrahydrofuroic acid, t-butyl ester (64 mg, from Reference Example 23, Step D) was dissolved in anhydrous methanol (1 mL) and cooled to xe2x88x9278xc2x0 C. Potassium bis(trimethylsilyl)amide 0.66 mL) was added dropwise over 10 minutes. After being stirred at 78xc2x0 C. for 15 minutes, trimethyl phosphonoacetate (65 mg, dissolved in 1.5 mL tetrahydrofuran) was added dropwise over 25 minutes. Upon completion of addition, the cold bath was removed and the mixture was allowed to stir at room temperature for 3 h when TLC (4/1 hexane/ethyl acetate) showed formation of a new spot. At 0xc2x0 C., the reaction was quenched by addition of 1.5 mL of saturated aqueous ammonium chloride and the product was extracted with ethyl acetate twice, washed with saturated sodium chloride and dried over anhydrous magnesium sulfate. The crude product obtained after filtration and removal of volatiles was flash chromatographed over silica get, using gradient elution (50-10/1 hexane/ethyl acetate) to give 31 mg of the desired compound, homogeneous by TLC (Rf=0.5 in 4/1 hexane/ethyl acetate). The NMR showed that all the diastereomers are present in this sample.
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.31, 1.39 (2d, J=7 Hz, 3H), 1.45, 1.47 (2s, 9H), 1.51, 1.56 (2s, 3H), 2.35-2.70 (m, 1H), 2.80-3.50(m, 1H), 3.68, 3.74(2s, 3H), 4.25(m, 1H), 5.9 (m, 1H).
Step B. 2,5-Dimethyl-3-methoxycarbonylmethyl-2-tetrahydrofuroic Acid, t-Butyl Ester
2,5-Dimethyl-3-methoxycarbonylmethylene-2-tetrahydrofuroic acid, t-butyl ester (28 mg, obtained from Step A) was dissolved in 0.25 mL of ethyl acetate and 0.25 mL of methanol. This mixture was hydrogenated under a balloon of hydrogen at room temperature for 1.25 h. The reaction mixture was filtered over a pad of celite, and the crude product obtained after removal of volatiles was purified via a silica gel SepPak, providing 21 mg of the desired compound cleanly (75% yield), homogeneous by TLC (Rf=0.2 in 10/1 hexane/ethyl acetate)
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.36(2d, J=7.2 Hz, 3H), 1.44 (s, 3H), 1.42 (m, 1H), 1.49 (s, 9H), 2.35-2.52 (m, 1H), 2.48-2.60(m, 1H), 3.70(s, 3H), 4.25(m, 1H).
Step C. 2.5-Dimethyl-3-methoxycarbonylmethyl-2-tetrahydrofuroic Acid
The title compound was obtained from 2,5-Dimethyl-3-methoxycarbonylmethyl-2-tetrahydrofuroic acid, t-butyl ester (Step B) according to the procedure of Example 122 Step B. The crude product was purified by a silica gel SepPak, homogeneous by TLC (Rf=0.2 in 1/1 hexane/ethyl acetate);
NMR: 500 MHz 1H NMR (CDCl3) xcex4 1.33(m, 1H), 1.39 (d, J=7.3 Hz, 3H), 1.53(s, 3H), 2.28 (m, 1H), 2.35(m, 1H), 2.65 (m, 1H), 2.75 (m, 1H), 3.70(s, 3H), 4.28(m, 1H).
Step A. N-(BOC)-4-[(Trifluoromethylsulfonyl)oxy]-(L)-phenylalanine, tert-Butyl Ester.
To a solution of N-(BOC)-(L)-tyrosine, tert-butyl ester (18.5 g, 55 mmol) in 150 mL of dry methylene chloride was added pyridine (17.4 g, 220 mmol) followed at 0xc2x0 C. by the dropwise addition of neat triflic anhydride (18.6 g , 66 mmol). The reaction mixture was stirred at 0xc2x0 C. and monitored by TLC. After 4 hours, the mixture was diluted with 200 mL of methylene chloride and was washed successively with 1N HCl (3xc3x97100 mL), saturated sodium bicarbonate (2xc3x97100 mL) and brine (1xc3x9750 mL). The solution was dried over anhydrous MgSO4, filtered and concentrated in vacuo to give N-(BOC)4-[(trifluoromethylsulfonyl)oxy]-(L)-phenylalanine, tert-butyl ester as an oil which was used without further purification.
Step B. N-(BOC)-(L)-4-(2xe2x80x2,6xe2x80x2-(Dimethoxyphenyl)phenylalanine, tert-Butyl Ester, Hydrochloride.
N-(BOC)4-[(trifluoromethylsulfonyl)oxy]-(L)-phenylalanine, tert-butyl ester (Step A) was dissolved in a mixture of 125 mL of toluene and 61 mL of ethanol. To this solution was added 2,6-dimethoxyboronic acid (11.3 g, 62 mmol) and palladium tetrakistriphenylphosphine (2.5 g). The solution was treated with of potassium carbonate (18.3 g, 133 mmol) dissolved in 30 mL of water. The mixture was heated to reflux over 4 hours, cooled to room temperature, and then diluted with 200 mL of ethyl acetate. The solution was washed with water (3xc3x9775 mL) and brine (1xc3x9775 mL) and was dried over anhydrous MgSO4. The mixture was filtered and concentrated in vacuo and the residue was purified by flash column chromatography on silica gel eluted with a gradient of 5-20% EtOAc in hexanes to provide 14.7 g of N-(BOC)-(L)-4-(2xe2x80x2,6xe2x80x2-(dimethoxyphenyl)phenylalanine, tert-butyl ester, hydrochloride as a white solid.
Step C. (L)-4-(2xe2x80x2,6xe2x80x2-(Dimethoxyphenyl)phenylalanine, tert-Butyl Ester Hydrochloride.
N-(BOC)-(L)-4-(2xe2x80x2,6xe2x80x2-(Dimethoxyphenyl)phenylalanine, tert-butyl ester, hydrochloride (Step B) was dissolved in 350 mL of tert-butyl acetate at 0xc2x0 C. and was treated with 8.3 mL of concentrated sulfuric acid. The cold bath was removed and after one hour TLC indicated only starting material was present. The reaction mixture was cooled in an ice bath once more and treated with 3.4 mL of concentrated sulfuric acid. The reaction was monitored by TLC. After consumption of the starting material the reaction mixture was diluted with 300 mL of ethyl acetate and was washed with 3xc3x97100 mL of 1N NaOH followed by brine (1xc3x97100 mL). The solution was dried over anhydrous MgSO4. Filtered and was concentrated in vacuo to provide 8.9 g of (L)-4-(2xe2x80x2,6xe2x80x2-(dimethoxyphenyl)phenylalanine, tert-butyl ester hydrochloride.
500 MHz 1H NMR (CD3OD): xcex4 1.45 (s, 9H), 3.20 (d, 2H); 3.69 (s, 6H); 4.20 (t, 1H); 6.72 (d, 2H), 7.15 (m, 5H).
Step A. 5-Chloro-2-(4-methoxyphenyl)-heptanoic Acid, Methyl Ester.
To a solution of 4-methoxymandelic acid methyl ester (1.1 g, 5.7 mmol) in 10 ml of THF was added lithium hexamethyldisilazane (12.5 ml, 1M in THF). The reaction was stirred at xe2x88x9278xc2x0 C. for 1.5 h, allowed to briefly warm to 0xc2x0 C., then cooled to xe2x88x9278xc2x0 C. 1-Bromo-3-chloropropane (1 g, 6.3 mmol) in 1 ml of THF was added to the solution and it was allowed to warm to room temperature. After stirring for 30 minutes, the reaction was quenched with water (25 ml) and the product extracted into ethyl acetate (50 ml). The organic layer was dried with magnesium sulfate, concentrated in vacuo then chromatographed over silica gel eluting with hexane/ethyl acetate (3:1) to 0.26 g of the product as a colorless oil.
500 MHz 1H NMR (CDCl3): 7.49 (d, J=8.9 Hz, 2H), 6.88 (d, J=9 Hz, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 3.54 (m, 2H), 2.22 (m, 2H), 1.8 (M, 2H).
Step B. 2-(4-Methoxyphenyl)-2-tetrahydrofuroic Acid, Methyl Ester
To a solution of 5-chloro-2-(4-methoxyphenyl)-heptanoic acid methyl ester (0.26 g, 0.96 mmol) in 5 ml of DMF at 0xc2x0 C. was added sodium hydride (0.6 g, 1.4 mmol). The reaction was stirred for 1 h at this temperature then quenched with 10 ml of water. The product was extracted into ethyl acetate (3xc3x9715 ml), washed with brine (20 ml), dried with magnesium sulfate and concentrated in vacuo to give a yellow oil. Silica gel chromatography eluting with hexane/ethyl acetate (4:1) gave 0.063 g of the title compound as a colorless oil.
500 MHz 1H NMR (CDCl3): 7.44 (d, J=8.9 Hz, 2H), 6.87 (d, J=8.6 Hz, 2H), 4.05 (m, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 2.78 (m, 1H), 2.18 (m, 1H), 1.95 (m, 2H).
Step C. 2-(4-methoxyphenyl)-2-tetrahydrofuroic Acid
To a solution of 2-(4-methoxyphenyl)-2-tetrahydrofuran Methyl Ester in 1 ml of methanol was added 5 drops of a 1N NaOH solution. After three hours, the reaction was quenched with 1 ml of 1N HCl and the product extracted into ethyl acetate (4xc3x971 ml). The organics were dried with magnesium sulfate and concentrated to give the title compound as a yellow solid (0.565 g).
2-(3,5-Dimethoxyphenyl)-2-tetrahydrofuroic acid was prepared according to the procedures described in Reference Example 27 substituting 3,5-dimethoxymandelic acid, methyl ester for 4-methoxymandelic acid, methyl ester in Step A.
Step A. N-2-hydroxyphenyl-2-tetrahydrofuroic Acetamide
To a solution of 2-tetrahydrofuroic acid (2.2 g, 19.2 mmol) and 2-aminophenol (1.0 g, 9.2 mmol) in 25 ml of methylene chloride was added PyBop (10.5 g, 22.9 mmol) and DIPEA (4.1 ml, 22.9 mmol). The reaction was stirred at room temperature for 20 h then diluted with ethyl acetate (50 ml). The organic layer was washed with water (50 ml), brine (50 ml) and dried with magnesium sulfate, concentrated in vacuo then chromatographed over silica gel eluting with hexane/ethyl acetate (6:4) to give 3.1 g of an oil. This oil was dissolved in 25 ml of toluene, treated with toluene sulfonic acid (1.9 g, 10.0 mmol) and heated to 75xc2x0 C. for 1 hr. The reaction was diluted with ethyl acetate (50 ml), dried with magnesium sulfate and concentrated to give a white solid 1.0 g.
500 MHz 1H NMR (CDCl3): 9.18 (s, 1H), 8.7 (s, 1H), 7.12 (m, 1H), 7.04 (m, 2H), 6.86 (m, 1H), 4.54 (m, 1H), 4.08 (m, 1H), 3.98 (m, 1H), 2.39, (m, 1H), 2.18 (m, 1H), 1.97 (m, 2H).
Step B. 2-(2-benzoxazole)-2-tetrahydrofuran
To a solution of N-2-hydroxyphenyl-2-tetrahydrofuroic acetamide (1.0 g, 5.0 mmol) and triphenylphosphine (1.95 g, 7.5 mmol) in 10 ml of THF was added DEAD (01.2 ml, 7.5 mmol). The reaction was stirred for 18 h at room temperature, diluted with ethyl acetate (50 ml), washed with water (50 ml), brine (50 ml), dried with magnesium sulfate, concentrated then chromatographed over silica gel eluting with hexane/ethyl acetate (2:1) to give 0.3 1 g of a colorless oil. 500 MHz 1H NMR (CDCl3): 7.7 (m, 1H), 7.5 (m, 1H), 7.3 (m, 2H), 5.2 (m, 1H), 4.1 (m, 1H), 4.0 (m, 1H), 2.4 (m, 2H), 2.15 (m, 1H), 2.05 (m, 1H).
Step C. 2-benzoxazole-2-tetrahydrofuroic Acid, Methyl Ester
To a solution of 2-benzoxazole-2-tetrahydrofuran (0.23 g, 1.19 mmol) in 2 ml of THF at xe2x88x9278xc2x0 C. was added methyl chloroformate (0.14 ml, 1.78 mmol) followed by LiHMDS (1.8 ml, 1M in THF). The reaction was stirred for 1 h at xe2x88x9278xc2x0 C. then quenched with 10 ml of water. The product was extracted into ethyl acetate (20 ml), washed with brine (10 ml), dried with magnesium sulfate, concentrated then chromatographed over silica gel eluting with hexane/ethyl acetate (3:1) to give 0.19 g of a colorless oil. 500 MHz 1H NMR (CDCl3): 7.75 (m, 1H), 7.54 (m, 1H), 7.36 (m, 2H), 4.22 (m, 1H), 4.25 (m, 1H), 3.8 (s, 3H), 3.08 (m, 1H), 2.62 (m, 1H), 2.16 (m, 2H).
Step A. 2-[(2-oxo-1-propyl)aminocarbonyl)]-2-tetrahydrofuroic Acid, tert-Butyl Ester.
To a solution of 2-carboxy-tetrahydrofuroic acid, tert-butyl ester (0.15 g, 0.069 mmol) and 1-amino-2-propanol (0.57 g, 0.076 mmol) was added PyBop (0.40 g, 0.076 mmol) and DIPEA (0.20 ml, 1.04 mmol). The reaction was stirred at room temperature for 17 h, concentrated in vacuo then chromatographed over silica gel eluting with hexane/ethyl acetate (3:1) to give 0.22 g of an oil. 0.24 g of this oil was oxidized to the corresponding ketone by treatment with cat. TPAP in the presence of excess NMO in methylene chloride for 1 hr. Filtration through silica gel eluted with hexane/ethyl acetate (1:1) resulted in 0.19 g of the title compound as a colorless oil. 500 MHz 1H NMR (CDCl3): 4.0-4.25 (m, 4H), 2.6 (m, 1H), 2.3 (m, 1H), 2.21 (s, 3H), 2.05 (m, 1H), 1.95 (m, 1H), 1.45 (s, 9H).
Step B. 2-(4-Methyl-2-thiazole)-2-tetrahydrofuroic Acid.
To a solution of (0.21 g, 0.079 mmol) in 0.25 ml toluene was added Lawesson""s Reagent (0.38 g, 0.095 mmol) and the reaction was heated to 100xc2x0 C. for 1 h. The reaction was concentrated then chromatographed over silica gel eluting with hexane/ethyl acetate (5:1) to give and oil. This oil was treated with trifluoroacetic acid in methylene chloride followed by silica gel chromatography eluting with methylene chloride/methanol/acetic acid (97:3:0.5) to give an oil (0.005 g). 500 MHz 1H NMR (CDCl3): 7.85 (s, 1H), 4.1-4.2 (m, 2H), 2.65 (m, 1H), 2.45 (m, 1H), 2.2 (m, 1H), 2.1 (s, 3H), 2.0 (m, 1H).
Step A. N-(BOC)-(L)-4-((2xe2x80x2,6xe2x80x2-dichloro)benzamido)-phenylalanine, Methyl Ester.
N(xcex1)-(BOC)-(L)-4-(FMOC-amino)-phenylalanine, methyl ester (9.62 g, 18.6 mmol) was dissolved in 15 mL of DMF and treated with diethylamine(11.6 mL, 112 mmol). The reaction mixture was stirred at room temperature for two hours, then concentrated in vacuo to give an viscous oil. This residue was dissolved in CH2Cl2 (50 mL) then treated with diisopropylethylamine (5.16 mL, 27.9 mmol) and 2,6-dichlorobenzoyl chloride (2.93 mL, 20.4 mmol). The reaction mixture was stirred overnight at room temperature and then quenched with H2O (40 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (2xc3x9740 mL). The combined organic layers were combined and washed with brine (1xc3x97200 mL) then dried over anhydrous MgSO4. The mixture was filtered and concentrated in vacuo, then the residue was purified by flash column chromatography eluted with 50% EtOAc in hexane to give N-(BOC)-(L)-4-((2xe2x80x2,6xe2x80x2-dichloro)benzamido)-phenylalanine, methyl ester (7.3 g).
500 MHz 1H NMR (CDCl3): 1.44 (s, 9H); 3.12 (m, 2H); 3.75 (s, 3H); 4.61 (m, 1H); 5.00 (d, 1H); 7.15 (d, 2H); 7.32 (m, 3H); 7.59 (d, 2H).
Step B (L)-4-((2xe2x80x2,6xe2x80x2-dichloro)benzamido)-phenylalanine, Methyl Ester Hydrochloride.
N-(BOC)-(L)-4-((2xe2x80x2,6xe2x80x2-dichloro)benzamido)-phenylalanine, methyl ester (2.50 g, 5.35 mmol) was dissolved in dioxane (5 mL) and treated with HCl in EtOAc (18.4 mL of 2.9 N). The mixture was stirred overnight at room temperature, then concentrated in vacuo to give a quantitative yield of (L)-4-((2xe2x80x2,6xe2x80x2-dichloro)-benzamido)phenylalanine, methyl ester hydrochloride.
500 MHz 1H NMR (CD3OD): 3.17 (m, 1H); 3.28 (m, 1H); 3.84 (s, 3H); 4.33 (m, 1H); 7.28 (d, 2H); 7.46 (m, 3H); 7.68 (d, 2H).
Step A. N-(Fmoc)-(L)-4-tert-Butoxycarbonyl-phenylalanine, Methyl Ester
To a solution of N-(9-fluorenylmethoxycarbonyl)-(L)-4-tert-butoxycarbonyl-phenylalanine (4.6 g, 9.4 mmol) in methylene chloride and methanol (50 mL each) at 0 C was added TMSCHN2 until a yellow color persisted (2 M, 15 mL, 14 mmol). After stirring at room temperature for 30 min, the mixture was concentrated to give the title compound (5.0 g) and was used without further purification.
Step B. N-(Fmoc)-(L)-4-Carboxy-phenylalanine, xcex1-Methyl Ester
To a solution of N-(Fmoc)-(L)-4-tert-butoxycarbonyl-phenylalanine, xcex1-methyl ester (5.0 g, 10 mmol) in 100 mL of methylene chloride was added trifluoroacetic acid (38 mL, 0.50 mol) mL at 0xc2x0 C. After stirring at room temperature overnight, the mixture was concentrated to give the title compound (4.4 g) and was used without further purification.
LC-MS: calculated for C26H23NO6, 445; found m/e 446 (M+H+).
Step C. N-(Fmoc)-(L)4-((4-N-tert-Butoxycarbonyl)piperazinyl-1-carbonyl)phenylalanine, xcex1-Methyl Ester
To a solution of N-(Fmoc)-(L)-4-carboxy-phenylalanine, xcex1-methyl ester (3.4 g, 7.6 mmol) and N-tert-butoxycarbonylpiperazine (1.4 g, 7.6 mmol) in 50 mL of methylene chloride at 0xc2x0 C. was added diisopropylethyl amine (2.7 mL, 15 mmol) and tris(pyrrolindinyl)phosphonium hexafluorophosphate (PyBOP, 4.2 mg, 8.0 mmol). After stirring at room temperature for 2 hr, TLC indicated complete consumption of the starting material. The reaction mixture was then concentrated, and the residue was purified on a silica gel column eluting with 1:4 acetone/hexane to give the title compound (4.0 g, 84%). LC-MS: calculated for C35H39N3O7, 613; found m/e 614 (M+H+).
Step D. (L)-4-((4-N-tert-Butoxycarbonyl)piperazinyl-1-carbonyl)phenylalanine, xcex1-Methyl Ester
To a solution of N-(Fmoc)-(L)4-((4-N-tert-butoxycarbonyl) piperazinyl-1-carbonyl)-phenylalanine, xcex1-methyl ester (4.0 g, 6.5 mmol) in methylene chloride (40 mL) was added diethyl amine (13 mL, 0.13 mol). After stirring at room temperature overnight, the reaction mixture was concentrated, and the residue was purified on a silica gel column eluting with methylene chloride to 1:20 methanol/methylene chloride to give the title compound (2.2 g, 89%). LC-MS: calculated for C20H29N3O5, 391; found m/e 392 (M+H+).
Step A. 3,5-Dimethoxybenzyl(tert-butyldiphenyl)silyly Ether
To 4.0 g (23.7 mmol) of 3,5-dimethoxybenzyl alcohol in 50 mL of DMF, 6.2 mL of tert-butyldiphenylsilyl chloride and 3.2 g (47.5 mmol) of imidazole were added. The reaction mixture was stirred overnight at 40xc2x0 C. The mixture was concentrated in vacuo to give a residue. The residue was purified by flash column chromatography on silica gel eluted with hexanes and 20% hexanes/ethyl acetate to afford a quantitative yield of the desired product. MS m/e=407.28 (M+H+).
Step B. 3,5-Dimethoxy-4-((tert-butyldiphenylsilyl)oxymethyl)phenylboronic Acid
To 9.3 g (22.8 mmol) of 3,5-dimethoxybenzyl(tert-butyldiphenyl)silyl ether in 25 mL of THF cooled to xe2x88x9278xc2x0 C., 16 mL of butyllithium (2.5 M in hexanes) was added. The mixture was stirred at xe2x88x9278xc2x0 C. for an hour, then warmed to room temperature and stirred for an hour. This mixture was cooled again to xe2x88x9278xc2x0 C. and 6.5 mL (57.1 mmol) of trimethyl borate was added. The mixture was warmed to room temperature and stirred for four hours. The mixture was quenched with 10 mL of water and continued to stir for a half hour. This mixture was acidified with acetic acid to pH 4. The mixture was extracted with ethyl acetate (3xc3x97). The organic layer was dried over anhydrous MgSO4. The mixture was filtered and concentrated in vacuo and the residue was purified by flash chromatography on silica gel eluted with 25% hexanes/ethyl acetate to give 3.7 g of the desired product. MS m/e=;451.41 (M+H+).
Step A. N-(2(R)-Methyl-2-tetrahydrofuroyl)-(L)-(4-(2,6-dimethoxy-4-tert-butyldiphenylsilyloxymethyl)phenyl)phenylalanine, Methyl Ester
N-(2(R)-Methyl-2-tetrahydrofuroyl)-(L)-4-iodophenylalanine, methyl ester (from Example 111, Step B) was coupled with 3,5-dimethoxy-4-((tert-butyldiphenylsilyloxymethyl)phenylboronic acid (from Reference Example 33, Step B) mediated by tetrakis-triphenylphosphine palladium(0) as described in Reference Example 2, Step B to provide 2.01 g of the desired product after flash column chromatography on silica gel eluted with 50% hexanes/ethyl acetate. MS m/e=696.36 (M+H+).
Step B. N-(2(R)-Methyl-2-tetrahydrofuroyl)-(L)-(4-(2,6-dimethoxy-4-hydroxymethyl)phenyl)phenylalanine, Methyl Ester
To 1.98 g (2.84 mmol) of N-(2(R)-methyl-2-tetrahydrofuroyl)-(L)-4-(2,6-dimethoxy-4-(tert-butyldiphenylsilyloxymethyl)phenyl)phenylalanine, methyl ester in 15 mL of THF cooled to 0xc2x0 C., 3.12 mL (3.12 mmol) of tetrabutylammonium fluoride (1.0 M in THF) was added. The mixture was stirred at 0xc2x0 C. for two hours. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel eluted with 50% hexanes/ethyl acetate to give 1.27 g of the desired product. MS m/e=458.32 (M+H+).
Step C. N-(2(R-Methyl-2-tetrahydrofuroyl)-(L)-(4-(2,6-dimethoxy-4-bromomethyl)phenyl)phenylalanine, Methyl Ester
To 2.52 g (5.97 mmol) of dibromotriphenylphosphine dissolved in 15 mL of CH2Cl2, a solution of 911 mg of N-(2(R)-methyl-2-tetrahydrofuroyl)-(L)-(4-(2,6-dimethoxy-4-hydroxymethyl)phenyl)phenylalanine, methyl ester was added. The reaction mixture was stirred overnight. The mixture was concentrated in vacuo and the residue was purified by flash column chromatography on silica gel eluted with 2:1 hexanes/ethyl acetate to give 692 mg of the desired product. MS m/e=522.1 (M+H+).